Agonistic anti-IL-2R antibodies and methods of use

ABSTRACT

Anti-IL2R (e.g., anti-IL2RB, anti-IL2RG, anti-IL2RB/G) antibodies are disclosed, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and use of such antibodies and compositions in the treatment of diseases and disorders that are mediated by the IL2/IL2R signaling pathway.

This application is a bypass continuation of International ApplicationNo. PCT/US2022/023058, filed Apr. 1, 2022, which claims the benefit ofpriority of U.S. Provisional Application No. 63/170,383 filed Apr. 2,2021, and U.S. Provisional Application No. 63/239,883 filed Sep. 1,2021, the contents of each of which are incorporated by reference hereinin their entireties.

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jan. 26, 2023, isnamed “TNO-0038-C1-US.xml” and is 103,397 bytes in size.

The present disclosure concerns antibodies that bind to theinterleukin-2 (IL-2) receptor (IL2R) and exhibit agonistic activity. Thedisclosure further concerns methods of making such antibodies,compositions, including pharmaceutical compositions, comprising suchantibodies, and the use of such antibodies and compositions in thetreatment of diseases and disorders that are mediated by the IL2/IL2Rsignaling pathway.

Despite having potent immune-activating activity and the potential toinduce durable tumor-regression in cancer patients, the success of IL-2as an immunotherapeutic has been limited by severe dose-limitingtoxicities. These adverse events are largely due to the preferentialuptake of IL-2 by cells that express the high-affinity, trimericreceptor, IL-2Rαβγ, such as T-regulatory (Treg) cells and endothelialcells. The present disclosure relates to antibodies (e.g.,multi-specific antibodies, multi-specific heavy-chain only antibodies,bispecific antibodies, bispecific heavy-chain only antibodies) that bindto and activate signaling through the dimeric IL-2Rβγ receptor complexthat is expressed on resting T-cells and NK cells. By avoiding bindingto IL-2Rα, the antibodies described herein eliminate the preferentialTreg activation of native IL-2, while maintaining potent stimulatoryeffects on other T-cell subsets along with NK cells. Additionally, thepresence of an Fc region on exemplary antibodies described hereinsignificantly extends the in vivo half-life over that of recombinantIL-2, permitting a more convenient dosing schedule in a therapeuticcontext. In vivo studies in both mice and cynomolgus monkeys haveconfirmed the in vivo biological activity, extended pharmacokinetics,and enhanced safety profile of exemplary antibodies described herein.Together, these results support the use of such antibodies as safe andeffective IL-2R agonists, as well as the use of the IL-2 signalingpathway as a therapeutic treatment approach for multiple types ofcancer.

The ability to harness the immune system against tumors has been firmlyestablished, with interleukin-2 (IL-2) being one of the firstrecombinant proteins to be successfully used as a treatment for cancernearly 40 years ago. Lotze et al., Journal of Immunology 135(4), 2865-75(1985); Rosenberg, S. A. J Immunol 192, 5451-58 (2014). IL-2 is a keyregulator of immune cells, inducing both T-cell and natural killer (NK)cell proliferation. However, IL-2 is a pleiotropic cytokine that alsoinduces the proliferation of immunosuppressive regulatory T (Treg)cells. Fontenot et al., Nat Immunol 6, 1142-51 (2005). The differentfunctions of IL-2 are determined by the composition of IL-2 receptorcomplex subunits expressed on different target cells. Boyman et al., NatRev Immunol 12, 180-90 (2012). The high affinity IL-2 receptor complexis composed of IL-2RA (CD25), IL-2RB (CD122), and the common gamma chainreceptor IL-2RG (CD132), and is expressed constitutively on CD4+FoxP3+Tregs cells and transiently on activated T cells. Waldmann, T. A., NatRev Immunol 6, 595-601 (2006). The intermediate affinity receptor iscomposed of only IL-2RB and IL-2RG, and is expressed on resting T-cells,CD8+ memory effector T-cells, and NK cells. Choudhry, H. et al, BiomedRes Int 2018, 1-7 (2018). The IL-2RA subunit is not required fordownstream JAK-STAT signaling, but its association with IL-2RB andIL-2RG provides a 100-fold higher affinity to IL-2 compared to theheterodimeric receptor composed only of IL-2RB and IL-2RG. Based onthese receptor binding differences and cell-specific expression, it hasbeen proposed that immunosuppressive Tregs serve as a buffer to consumelow levels of IL-2 and create a threshold effect for IL-2-mediatedexpansion of effector lymphocytes. Feinerman, O. et al., Mol Syst Biol6, 437 (2010).

Because of its unique signaling properties, low dose IL-2 has been usedclinically to stimulate Tregs to treat autoimmunity, while high-doseIL-2 was developed and approved (Proleukin®) for the treatment ofmetastatic melanoma and metastatic renal cell carcinoma, with durableresponses observed in 7-12% of patients. McDermott, D. F. et al., J ClinOncol 23, 133-141 (2004); Payne, R. et al., J Immunother Cancer 2, 13(2014); Atkins, M. B. et al., J Clin Oncol 17, 2105-2105 (1999);Rosenberg, S. A. et al., Ann Surg 228, 307-319 (1998). However, itsshort half-life and narrow therapeutic window have created significantchallenges for the safe and effective use of IL-2 in patients.Specifically, Proleukin® has severe side effects, including vascularleak syndrome, hypotension, and liver toxicities that have limited itsuse in cancer immunotherapy. It has been shown that the vascular leaktoxicity is related to the expression of the high affinity IL-2R onvascular endothelial cells and on lung endothelial cells, leading topulmonary edema. Krieg, C., et al., Proc National Acad Sci 107, 11906-11(2010). The anti-tumor effects of Proleukin® are further compromised byits preferential binding to the high affinity receptor on Treg cells,blunting its efficacy as an anti-cancer therapy. Schwartzentruber, D. J.et al., New Engl J Medicine 364, 2119-2127 (2011); Rezvani, K. et al.,Blood 108, 1291-1297 (2006). As an example, in melanoma patientsreceiving high dose IL-2 therapy, costimulator-positive (ICOS+) Tregcells were found to be the most proliferative lymphocyte population inthe blood after treatment with IL-2, and high numbers of ICOS+ Tregscorresponded with the worst patient outcomes. Sim, G. C. et al., J ClinInvest 124, 99-110 (2014).

Due to the pleiotropic nature of native IL-2 and its associatedlimitations as a therapeutic molecule, there has been substantial effortto engineer IL-2 variants that reduce dose-limiting toxicities andthereby broaden the therapeutic window. Murer, P. et al., New Biotechnol52, 42-53 (2019); Arenas-Ramirez, N. et al., Trends Immunol 36, 763-77(2015). Variant proteins that avoid the preferential activation ofhigh-affinity IL-2R-expressing cells, such as Tregs and vascularendothelial cells, are one approach to achieving this goal. In an effortto create such a molecule, other approaches have involved mutating theIL-2RA binding interface on IL-2, attaching polyethylene glycol to theIL-2 protein, creating synthetic IL-2 proteins, and generating anantibody that blocks the IL-2RA binding domain. Silva, D.-A. et al.,Nature 565, 186-191 (2019); Charych, D. H. et al., Clin Cancer Res 22,680-690 (2016); Arenas-Ramirez, N. et al., Sci Transl Med 8,367ra166-367ra166 (2016); Levin, A. M. et al., Nature 484, 529-533(2012); Lopes, J. E. et al., J Immunother Cancer 8, e000673 (2020). Asan alternative to IL-2, others have engineered IL-15 variants that bindto IL-2RB/IL-2RG subunits. The IL-15-specific receptor subunit naturallybinds to IL-15 in trans from antigen presenting cells; therefore, anactive IL-15 recombinant protein requires a single chain construct thatexpresses both IL-15 and the receptor subunit. Bernett, M. J. et al.Abstract 5565: Potency-reduced IL15/IL15Ra heterodimeric Fc-fusionsdisplay enhanced in vivo activity through increased exposure. 5565-5565(2018) doi:10.1158/1538-7445.am2018-5565. Mutated cytokines have alsobeen fused to antibodies or Fc domains to increase the in vivo half-lifeof the molecules and localize the cytokines to the tumor site. Murer, P.et al., New Biotechnol 52, 42-53 (2019); Klein, C. et al.,Oncoimmunology 6:3 e1277306 (2017); Schliemann, C. et al., Blood 120,3716-3716 (2012). While some of these engineered proteins have thedesired functional activity, many suffer from high levels ofimmunogenicity in vivo and present challenges with in vivo stability andmanufacturing. Brummelen, E. M. J. van et al., Oncotarget 9, 24737-49(2018); Groot, A. S. D. et al., Trends Immunol 28, 482-90 (2007);Schellekens, H., Nephrol Dial Transpl 18, 1257-59 (2003); Verhoef, J. J.F., et al., Drug Discov Today 19, 1945-52 (2014). Therefore, creating ananti-tumor agonist of the IL-2 pathway with the desired biologicalactivity, safety profile, and ideal drug-like properties remains asignificant challenge for the field.

The molecules of the present disclosure combine the favorable drug-likeproperties of antibodies with the functional behavior of molecules thatfacilitate IL-2RB and IL-2RG association and downstream signaling.Aspects of the disclosure include antibody sequences, such as fullyhuman antibody sequences, such as fully human multispecific (e.g.,bispecific) antibodies that simultaneously bind IL-2RB and IL-2RGsubunits and therefore mimic the activity of IL-2 while avoiding bindingto IL-2RA. In addition to exhibiting the desired activation andexpansion of immune effector cells, bispecific IL-2RB/G agonistantibodies described herein also avoid preferential expansion ofsuppressive Tregs, both in vitro and in vivo.

NON-LIMITING EXAMPLE EMBODIMENTS (SET 1)

Without limitation, some example embodiments/features of this disclosureinclude:

-   -   1. An antibody that binds to IL2RB, comprising a heavy chain        variable region comprising:        -   (a) a CDR1 sequence having two or fewer substitutions (e.g.,            0, 1, or 2) in any one of SEQ ID NOs: 1-3; and/or        -   (b) a CDR2 sequence having two or fewer substitutions (e.g.,            0, 1, or 2) in any one of SEQ ID NOs: 4-6; and/or        -   (c) a CDR3 sequence having two or fewer substitutions (e.g.,            0, 1, or 2) in any one of SEQ ID NOs: 7-10.    -   2. The antibody of Feature 1, comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3;            and/or        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6;            and/or        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.    -   3. The antibody of Feature 1 or 2, comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3;            and        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6;            and        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.    -   4. The antibody of any one of Features 1-3, comprising:        -   (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ            ID NO: 4, and a CDR3 sequence of SEQ ID NO: 7; or        -   (b) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ            ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8; or        -   (c) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ            ID NO: 5, and a CDR3 sequence of SEQ ID NO: 9; or        -   (d) a CDR1 sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ            ID NO: 6, and a CDR3 sequence of SEQ ID NO: 10.    -   5. The antibody of any one of Features 1-4, comprising a heavy        chain variable region having at least 95% sequence identity to        any one of SEQ ID NOs: 11-14.    -   6. The antibody of any one of Features 1-5, comprising a heavy        chain variable region sequence selected from SEQ ID NOs: 11-14.    -   7. An antibody that binds to IL2RB, comprising a heavy chain        variable region comprising:        -   (a) a CDR1 sequence comprising the formula:            -   G G S I S S S X1 W (SEQ ID NO:26)    -   where X1 is D or N;        -   (b) a CDR2 sequence comprising the formula:            -   IX2 H S G S T(SEQ ID NO: 27)    -   where X2 is D or S; and        -   (c) a CDR3 sequence comprising the formula:            -   X3 R G X4 W E L X5 D A F D I(SEQ ID NO: 28)    -   where X3 is G or A;        -   X4 is S or Q; and        -   X5 is S or T.    -   8. An antibody that binds to IL2RB, comprising a heavy chain        variable region comprising:        -   (a) a CDR1 sequence comprising the formula:            -   G F T F S X1 Y G(SEQ ID NO: 29)    -   where X1 is S or T;        -   (b) a CDR2 sequence comprising the formula:            -   I S Y D G S N X2 (SEQIDNO: 30)    -   where X2 is K or R; and        -   (c) a CDR3 sequence comprising the formula:            -   A R D L D Y D X3 L T G D P V G G F D I (SEQ ID NO: 31)    -   where X3 is V or I.    -   9. An antibody that binds to IL2RG, comprising a heavy chain        variable region comprising:        -   (a) a CDR1 sequence having two or fewer substitutions (e.g.,            0, 1, or 2) in any one of SEQ ID NOs: 15-16; and/or        -   (b) a CDR2 sequence having two or fewer substitutions (e.g.,            0, 1, or 2) in any one of SEQ ID NOs: 17-19; and/or        -   (c) a CDR3 sequence having two or fewer substitutions (e.g.,            0, 1, or 2) in any one of SEQ ID NOs: 20-21.    -   10. The antibody of Feature 9, comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16;            and/or        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19;            and/or        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.    -   11. The antibody of Feature 9 or 10, comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16;            and        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19;            and        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.    -   12. The antibody of any one of Features 9-11, comprising:        -   (a) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ            ID NO: 17, and a CDR3 sequence of SEQ ID NO: 20; or        -   (b) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ            ID NO: 18, and a CDR3 sequence of SEQ ID NO: 20; or        -   (c) a CDR1 sequence of SEQ ID NO: 16, a CDR2 sequence of SEQ            ID NO: 18, and a CDR3 sequence of SEQ ID NO: 20; or        -   (d) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ            ID NO: 19, and a CDR3 sequence of SEQ ID NO: 21.    -   13. The antibody of any one of Features 9-12, comprising a heavy        chain variable region having at least 95% sequence identity to        any one of SEQ ID NOs: 22-25.    -   14. The antibody of any one of Features 9-13, comprising a heavy        chain variable region sequence selected from SEQ ID NOs: 22-25.    -   15. An antibody that binds to IL2RG, comprising a heavy chain        variable region comprising:        -   (a) a CDR1 sequence comprising the formula: G F X1 X2 X3 X4            Y Y (SEQ ID NO: 32)    -   where X1 is T or I;        -   X2 is F or V;        -   X3 is S, N, or G; and        -   X4 is D or N;        -   (b) a CDR2 sequence comprising the formula:            -   I S X5 S G X6 X7 I (SEQ ID NO: 33)    -   where X5 is S or N;        -   X6 is D, S, G, or N; and        -   X7 is T or I; and        -   (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY            (SEQ ID NO: 20).    -   16. The antibody of any one of Features 1-15, wherein the CDR1,        CDR2, and CDR3 sequences are present in a human VH framework.    -   17. The antibody of any one of Features 1-16, wherein the        antibody is multi-specific.    -   18. The antibody of any one of Features 1-17, wherein the        antibody is bispecific.    -   19. The antibody of any one of Features 1-18, wherein the        antibody binds to IL2RB and IL2RG.    -   20. An antibody comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 7; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 17; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   21. The antibody of Feature 20, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        11, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 22.    -   22. The antibody of Feature 20 or 21, wherein the first heavy        chain variable region comprises SEQ ID NO: 11, and the second        heavy chain variable region comprises SEQ ID NO: 22.    -   23. The antibody of any one of Features 20-22, comprising a        first polypeptide comprising SEQ ID NO: 53 and a second        polypeptide comprising SEQ ID NO: 61.    -   24. An antibody comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 8; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 18; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   25. The antibody of Feature 24, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        12, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 23.    -   26. The antibody of Feature 24 or 25, wherein the first heavy        chain variable region comprises SEQ ID NO: 12, and the second        heavy chain variable region comprises SEQ ID NO: 23.    -   27. The antibody of any one of Features 24-26, comprising a        first polypeptide comprising SEQ ID NO: 62 and a second        polypeptide comprising SEQ ID NO: 63.    -   28. An antibody comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 2;            -   a CDR2 sequence of SEQ ID NO: 5; and            -   a CDR3 sequence of SEQ ID NO: 9; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 18; and            -   a CDR3 sequence of SEQ ID NO: 20; and    -   29. The antibody of Feature 28, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        13, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 23.    -   30. The antibody of Feature 28 or 29, wherein the first heavy        chain variable region comprises SEQ ID NO: 13, and the second        heavy chain variable region comprises SEQ ID NO: 23.    -   31. The antibody of any one of Features 28-30, comprising a        first polypeptide comprising SEQ ID NO: 64 and a second        polypeptide comprising SEQ ID NO: 65.    -   32. An antibody comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 3;            -   a CDR2 sequence of SEQ ID NO: 6; and            -   a CDR3 sequence of SEQ ID NO: 10; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 17; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   33. The antibody of Feature 32, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        14, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 22.    -   34. The antibody of Feature 32 or 33, wherein the first heavy        chain variable region comprises SEQ ID NO: 14, and the second        heavy chain variable region comprises SEQ ID NO: 22.    -   35. The antibody of any one of Features 32-34, comprising a        first polypeptide comprising SEQ ID NO: 66 and a second        polypeptide comprising SEQ ID NO: 67.    -   36. An antibody comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 8; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 16;            -   a CDR2 sequence of SEQ ID NO: 18; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   37. The antibody of Feature 36, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        12, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 24.    -   38. The antibody of Feature 36 or 37, wherein the first heavy        chain variable region comprises SEQ ID NO: 12, and the second        heavy chain variable region comprises SEQ ID NO: 24.    -   39. The antibody of any one of Features 36-38, comprising a        first polypeptide comprising SEQ ID NO: 34, and a second        polypeptide comprising SEQ ID NO: 35.    -   40. An antibody comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 8; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 19; and            -   a CDR3 sequence of SEQ ID NO: 21.    -   41. The antibody of Feature 40, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        12, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 25.    -   42. The antibody of Feature 40 or 41, wherein the first heavy        chain variable region comprises SEQ ID NO: 12, and the second        heavy chain variable region comprises SEQ ID NO: 25.    -   43. The antibody of any one of Features 40-42, comprising a        first polypeptide comprising SEQ ID NO: 36 and a second        polypeptide comprising SEQ ID NO: 37.    -   44. The antibody of any one of Features 20, 24, 28, 32, 36, or        40, wherein the CDR1, CDR2, and CDR3 sequences in the first        heavy chain variable region are present in a VH human framework.    -   45. The antibody of any one of Features 20, 24, 28, 32, 36, 40,        or 44, wherein the CDR1, CDR2, and CDR3 sequences in the second        heavy chain variable region are present in a VH human framework.    -   46. The antibody of any one of Features 1-22, 24-26, 28-30,        32-34, 36-38, 40-42, 44, or 45, wherein the antibody comprises        an Fc region.    -   47. The antibody of Feature 46, wherein the Fc region is a        variant Fc region.    -   48. The antibody of Feature 47, wherein the variant Fc region        possesses at least about 80% (e.g., at least about 85%, at least        about 90%, at least about 95%, at least about 98%, at least        about 99%) homology with a native sequence Fc region.    -   49. The antibody of Feature 47 or 48, wherein the variant Fc        region comprises heterodimerizing alterations.    -   50. The antibody of Feature 49, wherein the heterodimerizing        alterations comprise knob and holes substitutions (e.g., in a        variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the        other; 2) Y407A in one chain and T366W in the other; 3) F405A in        one chain and T394W in the other; 4) F405W in one chain and        T394S in the other; 5) Y407T in one chain and T366Y in the        other; 6) T366Y and F405A in one chain and T394W and Y407T in        the other; 7) T366W and F405W in one chain and T394S and Y407A        in the other; 8) F405W and Y407A in one chain and T366W and        T394S in the other; or 9) T366W in one polypeptide of the Fc and        T366S, L368A, and Y407V in the other).    -   51. The antibody of Feature 49 or 50, wherein the        heterodimerizing alterations comprise substitutions that create        new disulfide bridges (e.g., in a variant IgG1 Fc region, 1)        Y349C in one Fc polypeptide chain and S354C in the other; 2)        Y349C in one Fc polypeptide chain and E356C in the other; 3)        Y349C in one Fc polypeptide chain and E357C in the other; 4)        L351C in one Fc polypeptide chain and S354C in the other; 5)        T394C in one Fc polypeptide chain and E397C in the other; or 6)        D399C in one Fc polypeptide chain and K392C in the other).    -   52. The antibody of any one of Features 49-51, wherein the        heterodimerizing alterations comprise charge pair substitutions        (e.g., 1) K409E in one chain plus D399K in the other; 2) K409E        in one chain plus D399R in the other; 3) K409D in one chain plus        D399K in the other; 4) K409D in one chain plus D399R in the        other; 5) K392E in one chain plus D399R in the other; 6) K392E        in one chain plus D399K in the other; 7) K392D in one chain plus        D399R in the other; 8) K392D in one chain plus D399K in the        other; 9) K409D and K360D in one chain plus D399K and E356K in        the other; 10) K409D and K370D in one chain plus D399K and E357K        in the other; 11) K409D and K392D in one chain plus D399K,        E356K, and E357K in the other; 12) K409D and K392D on one chain        and D399K on the other; 13) K409D and K392D on one chain plus        D399K and E356K on the other; 14) K409D and K392D on one chain        plus D399K and D357K on the other; 15) K409D and K370D on one        chain plus D399K and D357K on the other; 16) D399K on one chain        plus K409D and K360D on the other; or 17) K409D and K439D on one        chain plus D399K and E356K on the other).    -   53. The antibody of any one of Features 47-52, wherein the        variant Fc region is a silenced Fc region.    -   54. The antibody of Feature 53, wherein the silenced Fc region        comprises substitution of one or more (e.g., two or more) of Fc        region residues 238, 265, 269, 270, 297, 327 and 329 according        to EU numbering.    -   55. The antibody of Feature 53 or 54, wherein the silenced Fc        region comprises a substitution that alters glycosylation.    -   56. The antibody of any one of Features 53-55, wherein the        silenced Fc region comprises an effector-less mutation (e.g., an        N297A, an N297G, a DANA mutation (D265A+N297A), or a DANG        mutation (D265A+N297G) in the CH2 region) and/or K322A and        L234A/L235A mutations.    -   57. The antibody of any one of Features 1-22, 24-26, 28-30,        32-34, 36-38, 40-42, or 44-56, wherein the antibody comprises a        heavy chain constant region sequence in the absence of a CH1        sequence.    -   58. The antibody of any one of Features 1-22, 24-26, 28-30,        32-34, 36-38, 40-42, or 44-57, wherein the antibody comprises a        heavy chain constant region comprising a hinge region, a CH2        domain, and a CH3 domain.    -   59. The antibody of Feature 58, wherein the hinge region        comprises a wild type human IgG4 hinge region sequence (SEQ ID        NO: 54).    -   60. The antibody of Feature 58, wherein the hinge region        comprises a variant human IgG4 hinge region sequence comprising        an S228P mutation (SEQ ID NO: 55).    -   61. The antibody of any one of Features 58-60, wherein the CH2        domain comprises a wild type human IgG4 CH2 domain sequence (SEQ        ID NO: 56).    -   62. The antibody of any one of Features 58-61, wherein the CH2        domain comprises a variant human IgG4 CH2 domain comprising an        F234A mutation, an L235A mutation, or both an F234A mutation and        an L235A mutation.    -   63. The antibody of any one of Features 58-62, wherein the CH3        domain comprises a wild type human IgG4 CH3 domain sequence (SEQ        ID NO: 58).    -   64. The antibody of any one of Features 58-62, wherein the CH3        domain comprises a variant human IgG4 CH3 domain sequence        comprising a T366W mutation.    -   65. The antibody of any one of Features 58-62, wherein the CH3        domain comprises a variant human IgG4 CH3 domain sequence        comprising a T366S, an L368A mutation, and a Y407V mutation.    -   66. The antibody of any one of Features 1-65, wherein the        antibody is a human antibody.    -   67. The antibody of any one of Features 1-66, wherein the        antibody is an isolated antibody.    -   68. The antibody of any one of Features 1-67, wherein the        antibody is an intact IgG molecule.    -   69. The antibody of any one of Features 1-68, wherein the        antibody is an intact IgG1 molecule.    -   70. The antibody of any one of Features 1-68, wherein the        antibody is an intact IgG2 molecule.    -   71. The antibody of any one of Features 1-68, wherein the        antibody is an intact IgG4 molecule.    -   72. The antibody of any one of Features 1-67, wherein the        antibody is an immunologically active portion of an intact IgG        molecule.    -   73. The antibody of any one of Features 1-67, wherein the        antibody is an immunologically active portion of an intact IgG1        molecule.    -   74. The antibody of any one of Features 1-67, wherein the        antibody is an immunologically active portion of an intact IgG2        molecule.    -   75. The antibody of any one of Features 1-67, wherein the        antibody is an immunologically active portion of an intact IgG4        molecule.    -   76. The antibody of any one of Features 1-67, wherein the        antibody is a triple-chain antibody-like molecule.    -   77. The antibody of any one of Features 1-67, wherein the        antibody is a heavy-chain only antibody.    -   78. The antibody of any one of Features 1-77, wherein the        antibody has a Tm of from about 55° C. to about 65° C.    -   79. The antibody of any one of Features 1-78, wherein the        antibody has a Tagg of from about 55° C. to about 65° C.    -   80. The antibody of any one of Features 1-79, wherein the        antibody has an affinity for IL2R with a Kd of from about 10⁻¹¹        M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M to around        about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; from        about 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to        around about 10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M;        from about 10⁻⁹ M to around about 10⁻⁸ M; from about 10⁻¹¹ M to        around about 10⁻⁹ M; from about 10⁻¹⁰ M to around about 10⁻⁹ M).    -   81. The antibody of any one of Features 1-80, wherein the        antibody has an affinity for IL2RB with a Kd of from about 10⁻⁸        M to around about 2.5×10⁻⁷ M.    -   82. The antibody of any one of Features 1-81, wherein the        antibody has an affinity for IL2RG with a Kd of from about 10⁻⁹        M to around about 2.5×10⁻⁸ M.    -   83. The antibody of any one of Features 80-82, wherein Kd is        measured using a ForteBio Octet Qk384 instrument in kinetics        mode.    -   84. The antibody of any one of Features 80-83, wherein Kd is        measured using a ForteBio Octet Qk384 instrument comprising an        anti-human Fc capture (AHC, 18-5005) sensor in kinetics mode.    -   85. The antibody of any one of Features 80-82, wherein Kd is        measured according to a method described in the Examples herein.    -   86. The antibody of any one of Features 1-85, wherein the        antibody functions as an IL2 receptor beta/gamma agonist.    -   87. A pharmaceutical composition comprising:        -   an antibody of any one of Features 1-86; and        -   a pharmaceutically acceptable excipient.    -   88. The pharmaceutical composition of Feature 87, wherein the        pharmaceutical composition is adapted for intravenous delivery.    -   89. The pharmaceutical composition of Feature 87, wherein the        pharmaceutical composition is adapted for subcutaneous delivery.    -   90. A polynucleotide encoding an antibody of any one of Features        1-86.    -   91. A vector comprising a polynucleotide of Feature 90.    -   92. A cell (e.g., a CHO cell) comprising a vector of Feature 91.    -   93. A method of producing an antibody of any one of Features        1-86, the method comprising:        -   growing a cell (e.g., a CHO cell) according to Feature 92            under conditions permissive for expression of the antibody;            and        -   isolating the antibody from the cell and/or a cell culture            medium in which the cell is grown.    -   94. A method of making an antibody of any one of Features 1-86,        the method comprising immunizing a transgenic animal (e.g., a        transgenic rat, a UniRat™ animal) with IL2R and identifying        IL2R-binding heavy chain sequences.    -   95. A kit for treating a disease or disorder in an individual in        need thereof comprising:        -   an antibody of any one of Features 1-86, or a pharmaceutical            composition of any one of Features 87-89; and        -   instructions for use.    -   96. The kit of Feature 95, further comprising at least one        additional reagent.    -   97. The kit of Feature 96, wherein the at least one additional        reagent comprises a chemotherapeutic drug.    -   98. A method of treating a disease or disorder comprising        administering to an individual in need thereof an effective dose        of an antibody of any one of Features 1-86, or a pharmaceutical        composition of any one of Features 87-89.    -   99. The method of Feature 98, wherein the antibody or        pharmaceutical composition is administered in conjunction with        another course of therapy.    -   100. The method of Feature 98 or 99, wherein the antibody or        pharmaceutical composition is administered in conjunction with a        chemotherapy regimen.    -   101. Use of an antibody of any one of Features 1-86 in the        preparation of a medicament for the treatment of a disease or        disorder in an individual in need thereof.    -   102. The use of Feature 101, wherein the medicament is intended        for administration in conjunction with another course of        therapy.    -   103. The use of Feature 101 or 102, wherein the medicament is        intended for administration in conjunction with a chemotherapy        regimen.    -   104. An antibody of any one of Features 1-86, or a        pharmaceutical composition of any one of Features 87-89, for use        in the treatment of a disease or disorder in an individual in        need thereof.    -   105. The antibody for use or pharmaceutical composition for use        of Feature 104, wherein the antibody or pharmaceutical        composition is intended for use in conjunction with another        course of therapy.    -   106. The antibody for use or pharmaceutical composition for use        of Feature 104, wherein the antibody or pharmaceutical        composition is intended for use in conjunction with a        chemotherapy regimen.    -   107. The kit, method, use, antibody for use, or pharmaceutical        composition for use of any one of Features 95-106, wherein the        disease or disorder is a cancer.    -   108. The kit, method, use, antibody for use, or pharmaceutical        composition for use of Feature 107, wherein the cancer is an        advanced or metastatic cancer.    -   109. The kit, method, use, antibody for use, or pharmaceutical        composition for use of Feature 107 or 108, wherein the cancer is        a solid tumor cancer.    -   110. The kit, method, use, antibody for use, or pharmaceutical        composition for use of Feature 109, wherein the solid tumor        cancer is selected from renal cell carcinoma, melanoma,        urothelial cancer, triple negative breast cancer, non-small cell        lung cancer (NSCLC), colorectal cancer, sarcoma, squamous cell        carcinoma of the head and neck, and metastatic        castration-resistant prostate cancer.    -   111. The kit, method, use, antibody for use, or pharmaceutical        composition for use of Feature 107 or 108, wherein the cancer is        a liquid cancer.    -   112. The kit, method, use, antibody for use, or pharmaceutical        composition for use of Feature 111, wherein the liquid cancer is        multiple myeloma or acute myeloid leukemia.    -   113. A method for stimulating IL2R signaling in an immune cell,        the method comprising contacting the immune cell with an        antibody of any one of Features 1-86, or a pharmaceutical        composition of any one of Features 87-89.    -   114. A method for stimulating an IL2RB/IL2RG dimeric receptor        complex on an immune cell, the method comprising contacting the        immune cell with an antibody of any one of Features 1-86, or a        pharmaceutical composition of any one of Features 87-89.    -   115. The method of Feature 113 or 114, wherein the immune cell        is selected from a CD4+ T-cell, a CD8+ T-cell, and a Natural        Killer (NK) cell.    -   116. Use of an antibody of any one of Features 1-86 in the        preparation of a medicament for stimulating IL2R signaling in an        immune cell in an individual in need thereof.    -   117. Use of an antibody of any one of Features 1-86 in the        preparation of a medicament for stimulating an IL2RB/IL2RG        dimeric receptor complex on an immune cell in an individual in        need thereof.    -   118. The use of Feature 116 or 117, wherein the immune cell is        selected from a CD4+ T-cell, a CD8+ T-cell, and a Natural Killer        (NK) cell.    -   119. An antibody of any one of Features 1-86, or a        pharmaceutical composition of any one of Features 87-89, for use        in a method for stimulating IL2R signaling in an immune cell.    -   120. An antibody of any one of Features 1-86, or a        pharmaceutical composition of any one of Features 87-89, for use        in a method for stimulating an IL2RB/IL2RG dimeric receptor        complex on an immune cell.    -   121. The antibody for use or pharmaceutical composition for use        of Feature 119 or 120, wherein the immune cell is selected from        a CD4+ T-cell, a CD8+ T-cell, and a Natural Killer (NK) cell.

NON-LIMITING EXAMPLE EMBODIMENTS (SET 2)

Without limitation, some example embodiments/clauses of this disclosureinclude:

-   -   1. A heavy chain-only antibody that binds to IL2RB, comprising a        heavy chain variable region comprising:        -   (a) a CDR1 sequence having two or fewer substitutions in any            one of SEQ ID NOs: 1-3; and/or        -   (b) a CDR2 sequence having two or fewer substitutions in any            one of SEQ ID NOs: 4-6; and/or        -   (c) a CDR3 sequence having two or fewer substitutions in any            one of SEQ ID NOs: 7-10.    -   2. The heavy chain-only antibody of Clause 1, wherein said CDR1,        CDR2, and CDR3 sequences are present in a human VH framework.    -   3. The heavy chain-only antibody of Clause 1 or 2, further        comprising a heavy chain constant region sequence in the absence        of a CH1 sequence.    -   4. The heavy chain-only antibody of any one of Clauses 1-3,        comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3;            and/or        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6;            and/or        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.    -   5. The heavy chain-only antibody of Clause 4, comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3;            and/or        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6;            and/or        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.    -   6. The heavy chain-only antibody of Clause 5, comprising:        -   (a) a CDR1 sequence of SEQ ID NO: 4, and a CDR3 sequence of            SEQ ID NO: 7; or        -   (b) a CDR1 sequence of SEQ ID NO: 4, and a CDR3 sequence of            SEQ ID NO: 8; or        -   (c) a CDR1 sequence of SEQ ID NO: 5, and a CDR3 sequence of            SEQ ID NO: 9; or        -   (d) a CDR1 sequence of SEQ ID NO: 6, and a CDR3 sequence of            SEQ ID NO: 10.    -   7. The heavy chain-only antibody of any one of Clauses 1-5,        comprising a heavy chain variable region having at least 95%        sequence identity to any one of SEQ ID NOs: 11-14.    -   8. The heavy chain-only antibody of Clause 7, comprising a heavy        chain variable region sequence selected from the group        consisting of SEQ ID NOs: 11-14.    -   9. A heavy chain-only antibody that binds to IL2RB, comprising a        heavy chain variable region comprising:        -   (a) a CDR1 sequence comprising the formula:            -   G G S I S S S X1 W (SEQ ID NO: 26)    -   where X1 is D or N;        -   (b) a CDR2 sequence comprising the formula:            -   I X2 H S G S T(SEQ ID NO: 27)    -   where X2 is D or S; and        -   (c) a CDR3 sequence comprising the formula:            -   X3 R G X4 W E L X5 D A F D I (SEQ ID NO: 28)    -   where X3 is G or A;        -   X4 is S or Q; and        -   X5 is S or T.    -   10. A heavy chain-only antibody that binds to IL2RB, comprising        a heavy chain variable region comprising:        -   (a) a CDR1 sequence comprising the formula:            -   G F T F S X1 Y G (SEQ ID NO: 29)    -   where X1 is S or T;        -   (b) a CDR2 sequence comprising the formula:            -   I S Y D G S N X2 (SEQ ID NO: 30)    -   where X2 is K or R; and        -   (c) a CDR3 sequence comprising the formula:            -   A R D L D Y D X3 L T G D P V G G F D I (SEQ ID NO: 31)    -   where X3 is V or I.    -   11. The heavy chain-only antibody of any one of Clauses 9-10,        wherein the CDR1, CDR2, and CDR3 sequences are present in a VH        human framework.    -   12. A heavy chain-only antibody that binds to IL2RG, comprising        a heavy chain variable region comprising:        -   (a) a CDR1 sequence having two or fewer substitutions in any            one of SEQ ID NOs: 15-16; and/or        -   (b) a CDR2 sequence having two or fewer substitutions in any            one of SEQ ID NOs: 17-19; and/or        -   (c) a CDR3 sequence having two or fewer substitutions in any            one of SEQ ID NOs: 20-21.    -   13. The heavy chain-only antibody of Clause 12, wherein said        CDR1, CDR2, and CDR3 sequences are present in a human VH        framework.    -   14. The heavy chain-only antibody of Clause 12 or 13, further        comprising a heavy chain constant region sequence in the absence        of a CH1 sequence.    -   15. The heavy chain-only antibody of any one of Clauses 12-14,        comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16;            and/or        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19;            and/or        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.    -   16. The heavy chain-only antibody of Clause 15, comprising:        -   (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16;            and/or        -   (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19;            and/or        -   (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.    -   17. The heavy chain-only antibody of Clause 16, comprising:        -   (a) a CDR1 sequence of SEQ ID NO: 15, a CDR2 SEQ ID NO: 17,            and a CDR3 sequence of SEQ ID NO: 20; or        -   (b) a CDR1 sequence of SEQ ID NO: 15, a CDR2 SEQ ID NO: 18,            and a CDR3 sequence of SEQ ID NO: 20; or        -   (c) a CDR1 sequence of SEQ ID NO: 16, a CDR2 SEQ ID NO: 18,            and a CDR3 sequence of SEQ ID NO: 20; or        -   (d) a CDR1 sequence of SEQ ID NO: 15, a CDR2 SEQ ID NO: 19,            and a CDR3 sequence of SEQ ID NO: 21.    -   18. The heavy chain-only antibody of any one of Clauses 12-16,        comprising a heavy chain variable region having at least 95%        sequence identity to any one of SEQ ID NOs: 22-25.    -   19. The heavy chain-only antibody of Clause 18, comprising a        heavy chain variable region sequence selected from the group        consisting of SEQ ID NOs: 22-25.    -   20. A heavy chain-only antibody that binds to IL2RG, comprising        a heavy chain variable region comprising:        -   (a) a CDR1 sequence comprising the formula:            -   G F X1 X2 X3 X4 Y Y (SEQ ID NO: 32)    -   where X1 is T or I;        -   X2 is F or V;        -   X3 is S, N, or G; and        -   X4 is D or N;        -   (b) a CDR2 sequence comprising the formula:            -   I S X5 S G X6 X7 I (SEQ ID NO: 33)    -   where X5 is S or N;        -   X6 is D, S, G, or N; and        -   X7 is T or I; and        -   (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY            (SEQ ID NO: 20).    -   21. The heavy chain-only antibody of Clause 20, wherein the        CDR1, CDR2, and CDR3 sequences are present in a VH human        framework.    -   22. The heavy chain-only antibody of any one of Clauses 1-21,        which is multi-specific.    -   23. The heavy chain-only antibody of Clause 22, which is        bispecific.    -   24. The heavy chain-only antibody of Clause 22 or 23, which        binds to IL2RB and IL2RG.    -   25. The heavy chain-only antibody of any one of Clauses 1-24,        further comprising a heavy chain constant region comprising a        hinge region, a CH2 domain, and a CH3 domain.    -   26. The heavy chain-only antibody of Clause 25, wherein the        hinge region comprises a wild type human IgG4 hinge region        sequence (SEQ ID NO: 54).    -   27. The heavy chain-only antibody of Clause 25, wherein the        hinge region comprises a variant human IgG4 hinge region        sequence comprising an S228P mutation (SEQ ID NO: 55).    -   28. The heavy chain-only antibody of any one of Clauses 25-27,        wherein the CH2 domain comprises a wild type human IgG4 CH2        domain sequence (SEQ ID NO: 56).    -   29. The heavy chain-only antibody of any one of Clauses 25-27,        wherein the CH2 domain comprises a variant human IgG4 CH2 domain        comprising an F234A mutation, an L235A mutation, or both an        F234A mutation and an L235A mutation.    -   30. The heavy chain-only antibody of any one of Clauses 25-29,        wherein the CH3 domain comprises a wild type human IgG4 CH3        domain sequence (SEQ ID NO: 58).    -   31. The heavy chain-only antibody of any one of Clauses 25-29,        wherein the CH3 domain comprises a variant human IgG4 CH3 domain        sequence comprising a T366W mutation.    -   32. The heavy chain-only antibody of any one of Clauses 25-29,        wherein the CH3 domain comprises a variant human IgG4 CH3 domain        sequence comprising a T366S, an L368A mutation, and a Y407V        mutation.    -   33. The heavy chain-only antibody of any one of Clauses 22-32,        which functions as an IL2 receptor beta/gamma agonist.    -   34. A bispecific agonistic anti-IL2R heavy chain-only antibody,        comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 7; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 17; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   35. The antibody of Clause 34, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        11, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 22.    -   36. The antibody of Clause 35, wherein the first heavy chain        variable region comprises SEQ ID NO: 11, and the second heavy        chain variable region comprises SEQ ID NO: 22.    -   37. The antibody of Clause 36, comprising a first polypeptide        comprising SEQ ID NO: 53 and a second polypeptide comprising SEQ        ID NO: 61.    -   38. A bispecific agonistic anti-IL2R heavy chain only antibody,        comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 8; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 18; and            -   a CDR3 sequence of SEQ ID NO: 20;    -   39. The antibody of Clause 38, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        12, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 23;    -   40. The antibody of Clause 39, wherein the first heavy chain        variable region comprises SEQ ID NO: 12, and the second heavy        chain variable region comprises SEQ ID NO: 23.    -   41. The antibody of Clause 40, comprising a first polypeptide        comprising SEQ ID NO: 62, and a second polypeptide comprising        SEQ ID NO: 63.    -   42. A bispecific agonistic anti-IL2R heavy chain-only antibody,        comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 2;            -   a CDR2 sequence of SEQ ID NO: 5; and            -   a CDR3 sequence of SEQ ID NO: 9; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 18; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   43. The antibody of Clause 42, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        13; and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 23.    -   44. The antibody of Clause 43, wherein the first heavy chain        variable region comprises SEQ ID NO: 13, and the second heavy        chain variable region comprises SEQ ID NO: 23.    -   45. The antibody of Clause 44, comprising a first polypeptide        comprising SEQ ID NO: 64 and a second polypeptide comprising SEQ        ID NO: 65.    -   46. A bispecific agonistic anti-IL2R heavy chain-only antibody,        comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 3;            -   a CDR2 sequence of SEQ ID NO: 6; and            -   a CDR3 sequence of SEQ ID NO: 10; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 17; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   47. The antibody of Clause 46, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        14, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 22.    -   48. The antibody of Clause 47, wherein the first heavy chain        variable region comprises SEQ ID NO: 14, and the second heavy        chain variable region comprises SEQ ID NO: 22.    -   49. The antibody of Clause 48, comprising a first polypeptide        comprising SEQ ID NO: 66 and a second polypeptide comprising SEQ        ID NO: 67.    -   50. A bispecific agonistic anti-IL2R heavy chain-only antibody,        comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 8; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 16;            -   a CDR2 sequence of SEQ ID NO: 18; and            -   a CDR3 sequence of SEQ ID NO: 20.    -   51. The antibody of Clause 50, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        12, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 24.    -   52. The antibody of Clause 51, wherein the first heavy chain        variable region comprises SEQ ID NO: 12, and the second heavy        chain variable region comprises SEQ ID NO: 24.    -   53. The antibody of Clause 52, comprising a first polypeptide        comprising SEQ ID NO: 34; and a second polypeptide comprising        SEQ ID NO: 35.    -   54. A bispecific agonistic anti-IL2R heavy chain-only antibody,        comprising:        -   a first heavy chain variable region that binds to IL2RB,            comprising:            -   a CDR1 sequence of SEQ ID NO: 1;            -   a CDR2 sequence of SEQ ID NO: 4; and            -   a CDR3 sequence of SEQ ID NO: 8; and        -   a second heavy chain variable region that binds to IL2RG,            comprising:            -   a CDR1 sequence of SEQ ID NO: 15;            -   a CDR2 sequence of SEQ ID NO: 19; and            -   a CDR3 sequence of SEQ ID NO: 21.    -   55. The antibody of Clause 54, wherein the first heavy chain        variable region has at least 95% sequence identity to SEQ ID NO:        12, and the second heavy chain variable region has at least 95%        sequence identity to SEQ ID NO: 25.    -   56. The antibody of Clause 55, wherein the first heavy chain        variable region comprises SEQ ID NO: 12, and the second heavy        chain variable region comprises SEQ ID NO: 25    -   57. The antibody of Clause 56, comprising a first polypeptide        comprising SEQ ID NO: 36 and a second polypeptide comprising SEQ        ID NO: 37.    -   58. A pharmaceutical composition comprising an antibody of any        one of Clauses 1-57.    -   59. A polynucleotide encoding an antibody of any one of Clauses        1-57.    -   60. A vector comprising the polynucleotide of Clause 59.    -   61. A cell comprising the vector of Clause 60.    -   62. A method of producing an antibody of any one of Clauses        1-57, the method comprising growing a cell according to Clause        61 under conditions permissive for expression of the antibody,        and isolating the antibody from the cell and/or a cell culture        medium in which the cell is grown.    -   63. A method of making an antibody of any one of Clauses 1-57,        the method comprising immunizing a UniRat animal with IL2R and        identifying IL2R-binding heavy chain sequences.    -   64. A kit for treating a disease or disorder in an individual in        need, comprising an antibody of any one of Clauses 1-57, or a        pharmaceutical composition of Clause 58, and instructions for        use.    -   65. The kit of Clause 64, further comprising at least one        additional reagent.    -   66. The kit of Clause 65, wherein the at least one additional        reagent comprises a chemotherapeutic drug.    -   67. A method of treatment, comprising administering to an        individual in need an effective dose of an antibody of any one        of Clauses 1-57, or a pharmaceutical composition of Clause 58.    -   68. Use of an antibody of any one of Clauses 1-57 in the        preparation of a medicament for the treatment of a disease or        disorder in an individual in need.    -   69. The antibody of any one of Clauses 1-57, or the        pharmaceutical composition of Clause 58, for use in therapy in        an individual in need.    -   70. A method for the treatment of a cancer, comprising        administering to a subject with said cancer an antibody of any        one of Clauses 1-57, or a pharmaceutical composition of Clause        58.    -   71. The method or use of any one of Clauses 67-70, wherein the        cancer is an advanced or metastatic cancer.    -   72. The method or use of any one of Clauses 67-71, wherein the        cancer is a solid tumor cancer.    -   73. The method or use of Clause 72, wherein the solid tumor        cancer is selected from the group consisting of: renal cell        carcinoma, melanoma, urothelial cancer, triple negative breast        cancer, non-small cell lung cancer (NSCLC), colorectal cancer,        sarcoma, squamous cell carcinoma of the head and neck, and        metastatic castration-resistant prostate cancer.    -   74. A method for stimulating IL2R signaling in an immune cell,        the method comprising contacting the immune cell with an        antibody of any one of Clauses 1-57, or a pharmaceutical        composition of Clause 58.    -   75. A method for stimulating an IL2RB/IL2RG dimeric receptor        complex on an immune cell, the method comprising contacting the        immune cell with an antibody of any one of Clauses 1-57, or a        pharmaceutical composition of Clause 58.    -   76. The method of Clause 74 or 75, wherein the immune cell is        selected from the group consisting of: a CD4+ T-cell, a CD8+        T-cell, and a Natural Killer (NK) cell.

IL-2R, also known as interleukin-2 receptor, is a heterodimeric proteinexpressed on the surface of various immune cells, which serves as acognate ligand for interleukin 2 (IL-2). The IL-2R complex is composedof various combinations of the IL-2Rα (ILR2A), IL-2Rβ (IL2RB), andIL-2Rγ (IL2RG) protein chains. IL-2RA is also referred to as CD25, andthe human IL2RA sequence (UniProtKB No. P01589) is provided herein asSEQ ID NO: 38. IL-2RB is also referred to as CD122, and the human IL2RBsequence (UniProtKB No. P14784) is provided herein as SEQ ID NO: 39.IL-2RG is also referred to as CD132, and the human IL2RG sequence(UniProtKB No. P31785) is provided herein as SEQ ID NO: 40. The humanIL-2 sequence (UniProtKB No. P60568) is provided herein as SEQ ID NO:41.

Aspects of the disclosure relate to an antibody that binds to IL2RB,comprising a heavy chain variable region comprising: (a) a CDR1 sequencehaving two or fewer (e.g., 0, 1, or 2) substitutions in any one of SEQID NOs: 1-3; and/or (b) a CDR2 sequence having two or fewer (e.g., 0, 1,or 2) substitutions in any one of SEQ ID NOs: 4-6; and/or (c) a CDR3sequence having two or fewer (e.g., 0, 1, or 2) substitutions in any oneof SEQ ID NOs: 7-10.

In some embodiments, the antibody comprises: (a) a CDR1 sequencecomprising any one of SEQ ID NOs: 1-3, and/or (b) a CDR2 sequencecomprising any one of SEQ ID NOs: 4-6; and/or (c) a CDR3 sequencecomprising any one of SEQ ID NOs: 7-10.

In some embodiments, the antibody comprises: (a) a CDR1 sequencecomprising any one of SEQ ID NOs: 1-3; and (b) a CDR2 sequencecomprising any one of SEQ ID NOs: 4-6; and (c) a CDR3 sequencecomprising any one of SEQ ID NOs: 7-10.

In some embodiments, the antibody comprises:

-   -   (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID        NO: 4, and a CDR3 sequence of SEQ ID NO: 7; or    -   (b) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID        NO: 4, and a CDR3 sequence of SEQ ID NO: 8; or    -   (c) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID        NO: 5, and a CDR3 sequence of SEQ ID NO: 9; or    -   (d) a CDR1 sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ ID        NO: 6, and a CDR3 sequence of SEQ ID NO: 10.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in ahuman VH framework.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having six or fewer (e.g., five or fewer,four or fewer, three or fewer, two or fewer; six, five, four, three,two, one, zero) substitutions in any one of SEQ ID NOs: 11-14. In someembodiments, the antibody comprises a heavy chain variable region havingat least 95% (e.g., at least 96%, at least 97%, at least 98%, at least99%) sequence identity to any one of SEQ ID NOs: 11-14. In someembodiments, the antibody comprises a heavy chain variable regionsequence selected from the group consisting of SEQ ID NOs: 11-14.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% homology(e.g., at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%) with a native-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody is multi-specific. In someembodiments, the antibody is bispecific. In some embodiments, theantibody binds to IL2RB and IL2RG. In some embodiments, the antibodyfunctions as an IL2 receptor beta/gamma agonist.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody that binds to IL2RB,comprising a heavy chain variable region comprising:

-   -   (a) a CDR1 sequence comprising the formula:        -   G G S I S S S X1 W (SEQ ID NO: 26)            where X1 is D or N;    -   (b) a CDR2 sequence comprising the formula:        -   I X2 H S G S T (SEQ ID NO: 27)            where X2 is D or S; and    -   (c) a CDR3 sequence comprising the formula: p2 X3 R G X4 W E L        X5 D A F D I (SEQ ID NO: 28)        where X3 is G or A; X4 is S or Q; and X5 is S or T.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody is multi-specific. In someembodiments, the antibody is bispecific. In some embodiments, theantibody binds to IL2RB and IL2RG. In some embodiments, the antibodyfunctions as an IL2 receptor beta/gamma agonist.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody that binds to IL2RB,comprising a heavy chain variable region comprising:

-   -   (a) a CDR1 sequence comprising the formula:        -   G F T F S X1 Y G (SEQ ID NO: 29)            where X1 is S or T;    -   (b) a CDR2 sequence comprising the formula:        -   I S Y D G S N X Z (SEQ ID NO: 30)            where X2 is K or R; and    -   (c) a CDR3 sequence comprising the formula:        -   A R D L D Y D X3 L T G D P V G G F D I (SEQ ID NO: 31)            where X3 is V or I.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody is multi-specific. In someembodiments, the antibody is bispecific. In some embodiments, theantibody binds to IL2RB and IL2RG. In some embodiments, the antibodyfunctions as an IL2 receptor beta/gamma agonist.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody that binds to IL2RG,comprising a heavy chain variable region comprising: (a) a CDR1 sequencehaving two or fewer (e.g., 0, 1, or 2) substitutions in any one of SEQID NOs: 15-16; and/or (b) a CDR2 sequence having two or fewer (e.g., 0,1, or 2) substitutions in any one of SEQ ID NOs: 17-19; and/or (c) aCDR3 sequence having two or fewer (e.g., 0, 1, or 2) substitutions inany one of SEQ ID NOs: 20-21.

In some embodiments, the antibody comprises: (a) a CDR1 sequencecomprising any one of SEQ ID NOs: 15-16; and/or (b) a CDR2 sequencecomprising any one of SEQ ID NOs: 17-19; and/or (c) a CDR3 sequencecomprising any one of SEQ ID NOs: 20-21.

In some embodiments, the antibody comprises: (a) a CDR1 sequencecomprising any one of SEQ ID NOs: 15-16; and (b) a CDR2 sequencecomprising any one of SEQ ID NOs: 17-19; and (c) a CDR3 sequencecomprising any one of SEQ ID NOs: 20-21.

In some embodiments, the antibody comprises:

-   -   (a) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID        NO: 17, and a CDR3 sequence of SEQ ID NO: 20; or    -   (b) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID        NO: 18, and a CDR3 sequence of SEQ ID NO: 20; or (c) a CDR1        sequence of SEQ ID NO: 16, a CDR2 sequence of SEQ ID NO: 18, and        a CDR3 sequence of SEQ ID NO: 20; or    -   (d) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID        NO: 19, and a CDR3 sequence of SEQ ID NO: 21.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having six or fewer (e.g., five or fewer,four or fewer, three or fewer, two or fewer; six, five, four, three,two, one, zero) substitutions in any one of SEQ ID NOs: 22-25. In someembodiments, the antibody comprises a heavy chain variable region havingat least 95% (e.g., at least 96%, at least 97%, at least 98%, at least99%) sequence identity to any one of SEQ ID NOs: 22-25. In someembodiments, the antibody comprises a heavy chain variable regionsequence selected from the group consisting of SEQ ID NOs: 22-25.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody is multi-specific. In someembodiments, the antibody is bispecific. In some embodiments, theantibody binds to IL2RB and IL2RG. In some embodiments, the antibodyfunctions as an IL2 receptor beta/gamma agonist.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody that binds to IL2RG,comprising a heavy chain variable region comprising:

-   -   (a) a CDR1 sequence comprising the formula:        -   G F X1 X2 X3 X4 Y Y (SEQ ID NO: 32)            where X1 is T or I; X2 is F or V; X3 is S, N, or G; and X4            is D or N;    -   (b) a CDR2 sequence comprising the formula:        -   I S X5 S G X6 X7 I (SEQ ID NO: 33)            where X5 is S or N; X6 is D, S, G, or N; and X7 is T or I;            and    -   (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID        NO: 20).

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, the antibody is multi-specific. In someembodiments, the antibody is bispecific. In some embodiments, theantibody binds to IL2RB and IL2RG.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody is an Fc-region-containing antibody.In some embodiments, the antibody further comprises a heavy chainconstant region comprising a hinge region, a CH2 domain, and a CH3domain. In some embodiments, the heavy chain constant region does notcontain a CH1 sequence. In some embodiments, the hinge region comprisesa wild type human IgG4 hinge region sequence (SEQ ID NO: 54). In someembodiments, the hinge region comprises a variant human IgG4 hingeregion sequence comprising an S228P mutation (SEQ ID NO: 55). In someembodiments, the CH2 domain comprises a wild type human IgG4 CH2 domainsequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprisesa variant human IgG4 CH2 domain comprising an F234A mutation, an L235Amutation, or both an F234A mutation and an L235A mutation. In someembodiments, the CH3 domain comprises a wild type human IgG4 CH3 domainsequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprisesa variant human IgG4 CH3 domain sequence comprising a T366W mutation. Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366S, an L368A mutation, and a Y407Vmutation.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody functions as an IL2 receptorbeta/gamma agonist.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of SEQ ID NO: 4; and aCDR3 sequence of SEQ ID NO: 7; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 17; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 11, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 22. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 11, and thesecond heavy chain variable region comprises SEQ ID NO: 22.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54) In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody comprises a first polypeptidecomprising SEQ ID NO: 53 and a second polypeptide comprising SEQ ID NO:61.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of SEQ ID NO: 4; and aCDR3 sequence of SEQ ID NO: 8; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 18; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 12, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 12, and thesecond heavy chain variable region comprises SEQ ID NO: 23.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody comprises a first polypeptidecomprising SEQ ID NO: 62 and a second polypeptide comprising SEQ ID NO:63.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻⁸ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of of SEQ ID NO: 2; a CDR2 sequence of SEQ ID NO: 5; and aCDR3 sequence of SEQ ID NO: 9; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 18; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 13, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 13, and thesecond heavy chain variable region comprises SEQ ID NO: 23.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody comprises a first polypeptidecomprising SEQ ID NO: 63 and a second polypeptide comprising SEQ ID NO:65.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 3; a CDR2 sequence of SEQ ID NO: 6; and aCDR3 sequence of SEQ ID NO: 10; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 17; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 14, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 22. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 14; and thesecond heavy chain variable region comprises SEQ ID NO: 22.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody comprises a first polypeptidecomprising SEQ ID NO: 66 and a second polypeptide comprising SEQ ID NO:67.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody, comprising:

-   -   a first heavy chain variable region that binds to IL2RB,        comprising: a CDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of        SEQ ID NO: 4; and a CDR3 sequence of SEQ ID NO: 8; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 16; a CDR2 sequence of SEQ ID NO: 18; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 12, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 24. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 12, and thesecond heavy chain variable region comprises SEQ ID NO: 24.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody comprises a first polypeptidecomprising SEQ ID NO: 34 and a second polypeptide comprising SEQ ID NO:35.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include an antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of SEQ ID NO: 4; and aCDR3 sequence of SEQ ID NO: 8; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 19; and aCDR3 sequence of SEQ ID NO: 21.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 12, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 25. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 12, and thesecond heavy chain variable region comprises SEQ ID NO: 25.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibody is a human antibody. In some embodiments, theantibody is an isolated human antibody.

In some embodiments, the antibody is an intact IgG molecule. In someembodiments, the antibody is an intact IgG1 molecule. In someembodiments, the antibody is an intact IgG2 molecule. In someembodiments, the antibody is an intact IgG4 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG1 molecule. In someembodiments, the antibody is an immunologically active portion of anintact IgG2 molecule. In some embodiments, the antibody is animmunologically active portion of an intact IgG4 molecule. In someembodiments, the antibody is a triple-chain antibody-like molecule. Insome embodiments, the antibody is a heavy-chain only antibody.

In some embodiments, the antibody comprises a Fc region. In someembodiments, the antibody comprises a variant Fc region. In someembodiments, the variant Fc region possesses at least about 80% (e.g.,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%) homology with a native-sequence Fcregion.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, the antibody further comprises a heavy chainconstant region sequence in the absence of a CH1 sequence. In someembodiments, the antibody comprises a heavy chain constant regioncomprising a hinge region, a CH2 domain, and a CH3 domain. In someembodiments, the hinge region comprises a wild type human IgG4 hingeregion sequence (SEQ ID NO: 54). In some embodiments, the hinge regioncomprises a variant human IgG4 hinge region sequence comprising an S228Pmutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprisesa wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In someembodiments, the CH2 domain comprises a variant human IgG4 CH2 domaincomprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, the antibody comprises a first polypeptidecomprising SEQ ID NO: 36 a nd a second polypeptide comprising SEQ ID NO:37.

In some embodiments, the antibody has a Tm of from about 55° C. to about65° C. In some embodiments, the antibody has a Tagg of from about 55° C.to about 65° C. In some embodiments, the antibody has a Tm of from about55° C. to about 65° C. and a Tagg of from about 55° C. to about 65° C.

In some embodiments, the antibody has an affinity for IL2R with a Kd offrom about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M toaround about 10⁻⁶ M; from about 10⁻⁹ M to around about 10⁻⁶ M; fromabout 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M to around about10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M toaround about 10⁻⁸ M; from about 10⁻¹¹ M to around about 10⁻⁹ M; fromabout 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, the antibody has an affinity for IL2RG with a Kd offrom about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the antibody has an affinity for IL2RB with a Kd offrom about 10⁻⁸ M to around about 2.5×10⁻⁷ M and an affinity for IL2RGwith a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

In a conventional IgG antibody, the association of the heavy chain andthe light chain is due in part to a hydrophobic interaction between thelight chain constant region and the CH1 constant domain of the heavychain. There are additional residues in the heavy chain framework 2(FR2) and framework 4 (FR4) regions that also contribute to thishydrophobic interaction between the heavy and light chains.

It is known, however, that sera of camelids (sub-order Tylopoda whichincludes camels, dromedaries, and llamas) contain a major type ofantibodies composed solely of paired H-chains (heavy-chain onlyantibodies or HCAbs). The heavy-chain only antibodies of Camelidae(Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lamaalpaca and Lama vicugna) have a unique structure consisting of a singlevariable domain (VHH), a hinge region, and two constant domains (CH2 andCH3), which are highly homologous to the CH2 and CH3 domains ofclassical antibodies. These heavy-chain only antibodies lack the firstdomain of the constant region (CH1), which is present in the genome butis spliced out during mRNA processing. The absence of the CH1 domainexplains the absence of the light chain in the heavy-chain onlyantibodies since this domain is the anchoring place for the constantdomain of the light chain. Such heavy-chain only antibodies naturallyevolved to confer antigen-binding specificity and high affinity by threeCDRs from conventional antibodies or fragments thereof. Muyldermans,2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin BiolTher 5:111-124. Cartilaginous fish, such as sharks, have also evolved adistinctive type of immunoglobulin, designated as IgNAR, which lacks thelight polypeptide chains and is composed entirely by heavy chains. IgNARmolecules can be manipulated by molecular engineering to produce thevariable domain of a single heavy chain polypeptide (vNARs). Nuttall etal. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function andBioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology40, 25-33 (2003).

The ability of heavy chain-only antibodies devoid of light chain to bindantigen was established in the 1960s (Jaton et al. (1968) Biochemistry,7, 4185-4195). Heavy chain immunoglobulin physically separated fromlight chain retained 80% of antigen-binding activity relative to thetetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstratedthat removal of the CH1 domain from a rearranged mouse μ gene results inthe production of a heavy chain-only antibody, devoid of light chain, inmammalian cell culture. The antibodies produced retained VH bindingspecificity and effector functions.

Heavy chain antibodies with high specificity and affinity can begenerated against a variety of antigens through immunization (van derLinden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), andthe VHH portion can be readily cloned and expressed in yeast (Frenken,L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels ofexpression, solubility and stability are significantly higher than thoseof classical F(ab) or Fv fragments. Ghahroudi, M. A. et al. FEBS Lett.414, 521-526 (1997).

Mice in which the λ (lambda) light (L) chain locus and/or the λ and κ(kappa) L chain loci have been functionally silenced and antibodiesproduced by such mice are described in U.S. Pat. Nos. 7,541,513 and8,367,888. Recombinant production of heavy chain-only antibodies in miceand rats has been reported, for example, in WO2006008548; U.S.Application Publication No. 20100122358; Nguyen et al., 2003,Immunology; 109(1), 93-101; Brüggemann et al., Crit. Rev. Immunol.;2006, 26(5):377-90; and Zou et al., 2007, J Exp Med; 204(13): 3271-3283.The production of knockout rats via embryo microinjections ofzinc-finger nucleases is described in Geurts et al., 2009, Science,325(5939):433. Soluble heavy chain-only antibodies and transgenicrodents comprising a heterologous heavy chain locus producing suchantibodies are described in U.S. Pat. Nos. 8,883,150 and 9,365,655.CAR-T structures comprising single-domain antibodies as binding(targeting) domains are described, for example, in Iri-Sofla et al.,2011, Experimental Cell Research 317:2630-2641, and Jamnani et al.,2014, Biochim Biophys Acta, 1840:378-386.

Aspects of the disclosure include heavy chain-only antibodies that bindto IL2RB, comprising a heavy chain variable region comprising: (a) aCDR1 sequence having two or fewer (e.g., 0, 1, or 2) substitutions inany one of SEQ ID NOs: 1-3; and/or (b) a CDR2 sequence having two orfewer (e.g., 0, 1, or 2) substitutions in any one of SEQ ID NOs: 4-6;and/or (c) a CDR3 sequence having two or fewer (e.g., 0, 1, or 2)substitutions in any one of SEQ ID NOs: 7-10.

In some embodiments, a heavy chain-only antibody comprises: (a) a CDR1sequence comprising any one of SEQ ID NOs: 1-3; and/or (b) a CDR2sequence comprising any one of SEQ ID NOs: 4-6; and/or (c) a CDR3sequence comprising any one of SEQ ID NOs: 7-10.

In some embodiments, a heavy chain-only antibody comprises: (a) a CDR1sequence comprising any one of SEQ ID NOs: 1-3; and (b) a CDR2 sequencecomprising any one of SEQ ID NOs: 4-6; and (c) a CDR3 sequencecomprising any one of SEQ ID NOs: 7-10.

In some embodiments, a heavy chain-only antibody comprises:

-   -   (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID        NO: 4, and a CDR3 sequence of SEQ ID NO: 7; or    -   (b) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID        NO: 4, and a CDR3 sequence of SEQ ID NO: 8, or    -   (c) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID        NO: 5, and a CDR3 sequence of SEQ ID NO: 9, or    -   (d) a CDR1 sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ ID        NO: 6, and a CDR3 sequence of SEQ ID NO: 10.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in ahuman VH framework.

In some embodiments, a heavy chain-only antibody comprises a heavy chainvariable region comprising a sequence having six or fewer (e.g., five orfewer, four or fewer, three or fewer, two or fewer; six, five, four,three, two, one, zero) substitutions in any one of SEQ ID NOs: 11-14. Insome embodiments, a heavy chain-only antibody comprises a heavy chainvariable region having at least 95% (e.g., at least 96%, at least 97%,at least 98%, at least 99%) sequence identity to any one of SEQ ID NOs:11-14. In some embodiments, a heavy chain-only antibody comprises aheavy chain variable region sequence selected from the group consistingof SEQ ID NOs: 11-14.

In some embodiments, a heavy-chain only antibody comprises a Fc region.In some embodiments, the heavy-chain only antibody comprises a variantFc region. In some embodiments, the variant Fc region possesses at leastabout 80% homology (e.g., at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%) with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a heavy chain-only antibody further comprises aheavy chain constant region sequence in the absence of a CH1 sequence.In some embodiments, the heavy chain-only antibody comprises a heavychain constant region comprising a hinge region, a CH2 domain, and a CH3domain. In some embodiments, the hinge region comprises a wild typehuman IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments,the hinge region comprises a variant human IgG4 hinge region sequencecomprising an S228P mutation (SEQ ID NO: 55). In some embodiments, theCH2 domain comprises a wild type human IgG4 CH2 domain sequence (SEQ IDNO: 56). In some embodiments, the CH2 domain comprises a variant humanIgG4 CH2 domain comprising an F234A mutation, an L235A mutation, or bothan F234A mutation and an L235A mutation. In some embodiments, the CH3domain comprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO:58). In some embodiments, the CH3 domain comprises a variant human IgG4CH3 domain sequence comprising a T366W mutation. In some embodiments,the CH3 domain comprises a variant human IgG4 CH3 domain sequencecomprising a T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a heavy chain-only antibody has a Tm of from about55° C. to about 65° C. In some embodiments, a heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, aheavy chain-only antibody has a Tm of from about 55° C. to about 65° C.and a Tagg of from about 55° C. to about 65° C.

In some embodiments, a heavy chain-only antibody is multi-specific. Insome embodiments, a heavy chain-only antibody is bispecific. In someembodiments, a heavy chain-only antibody binds to IL2RB and IL2RG. Insome embodiments, a heavy chain-only antibody functions as an IL2receptor beta/gamma agonist.

In some embodiments, a heavy chain-only antibody has an affinity forIL2R with a Kd of from about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., fromabout 10⁻¹⁰ M to around about 10⁻⁶ M; from about 10⁻⁹ M to around about10⁻⁶ M; from about 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M toaround about 10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; fromabout 10⁻⁹ M to around about 10⁻⁸ M; from about 10⁻¹¹ M to around about10⁻⁹ M; from about 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RG with a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M and anaffinity for IL2RG with a Kd of from about 10⁻⁹ M to around about2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include heavy chain-only antibodies that bindto IL2RB, comprising a heavy chain variable region comprising:

-   -   (a) a CDR1 sequence comprising the formula:        -   G G S I S S S X1 W (SEQ ID NO: 26)            where X1 is D or N;    -   (b) a CDR2 sequence comprising the formula:        -   I X2 H S G S T (SEQ ID NO: 27)            where X2 is D or S; and    -   (c) a CDR3 sequence comprising the formula:        -   X3 R G X4 W E L X5 D A F D I (SEQ ID NO: 28)            where X3 is G or A; X4 is S or Q; and X5 is S or T.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, a heavy-chain only antibody comprises a Fc region.In some embodiments, the heavy-chain only antibody comprises a variantFc region. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a heavy chain-only antibody further comprises aheavy chain constant region sequence in the absence of a CH1 sequence.In some embodiments, the heavy chain-only antibody comprises a heavychain constant region comprising a hinge region, a CH2 domain, and a CH3domain. In some embodiments, the hinge region comprises a wild typehuman IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments,the hinge region comprises a variant human IgG4 hinge region sequencecomprising an S228P mutation (SEQ ID NO: 55). In some embodiments, theCH2 domain comprises a wild type human IgG4 CH2 domain sequence (SEQ IDNO: 56). In some embodiments, the CH2 domain comprises a variant humanIgG4 CH2 domain comprising an F234A mutation, an L235A mutation, or bothan F234A mutation and an L235A mutation. In some embodiments, the CH3domain comprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO:58). In some embodiments, the CH3 domain comprises a variant human IgG4CH3 domain sequence comprising a T366W mutation. In some embodiments,the CH3 domain comprises a variant human IgG4 CH3 domain sequencecomprising a T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a heavy chain-only antibody has a Tm of from about55° C. to about 65° C. In some embodiments, a heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, aheavy chain-only antibody has a Tm of from about 55° C. to about 65° C.and a Tagg of from about 55° C. to about 65° C.

In some embodiments, a heavy chain-only antibody is multi-specific. Insome embodiments, a heavy chain-only antibody is bispecific. In someembodiments, a heavy chain-only antibody binds to IL2RB and IL2RG. Insome embodiments, a heavy chain-only antibody functions as an IL2receptor beta/gamma agonist.

In some embodiments, a heavy chain-only antibody has an affinity forIL2R with a Kd of from about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., fromabout 10⁻¹⁰ M to around about 10⁻⁶ M; from about 10⁻⁹ M to around about10⁻⁶ M; from about 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M toaround about 10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; fromabout 10⁻⁹ M to around about 10⁻⁸ M; from about 10⁻¹¹ M to around about10⁻⁹ M; from about 10⁻¹¹ M to around about 10⁻⁹ M).

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁷ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RG with a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M and anaffinity for IL2RG with a Kd of from about 10⁻⁹ M to around about2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include heavy chain-only antibodies that bindto IL2RB, comprising a heavy chain variable region comprising:

-   -   (a) a CDR1 sequence comprising the formula:        -   G F T F S X1 Y G (SEQ ID NO: 29)            where X1 is S or T;    -   (b) a CDR2 sequence comprising the formula:        -   I S Y D G S N X2 (SEQ ID NO: 30)            where X2 is K or R; and    -   (c) a CDR3 sequence comprising the formula:        -   A R D L D Y D X3 L T G D P V G G F D I (SEQ ID NO: 31)            where X3 is V or I.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, a heavy-chain only antibody comprises a Fc region.In some embodiments, the heavy-chain only antibody comprises a variantFc region. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a heavy chain-only antibody further comprises aheavy chain constant region sequence in the absence of a CH1 sequence.In some embodiments, the heavy chain-only antibody comprises a heavychain constant region comprising a hinge region, a CH2 domain, and a CH3domain. In some embodiments, the hinge region comprises a wild typehuman IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments,the hinge region comprises a variant human IgG4 hinge region sequencecomprising an S228P mutation (SEQ ID NO: 55). In some embodiments, theCH2 domain comprises a wild type human IgG4 CH2 domain sequence (SEQ IDNO: 56). In some embodiments, the CH2 domain comprises a variant humanIgG4 CH2 domain comprising an F234A mutation, an L235A mutation, or bothan F234A mutation and an L235A mutation. In some embodiments, the CH3domain comprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO:58). In some embodiments, the CH3 domain comprises a variant human IgG4CH3 domain sequence comprising a T366W mutation. In some embodiments,the CH3 domain comprises a variant human IgG4 CH3 domain sequencecomprising a T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a heavy chain-only antibody has a Tm of from about55° C. to about 65° C. In some embodiments, a heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, aheavy chain-only antibody has a Tm of from about 55° C. to about 65° C.and a Tagg of from about 55° C. to about 65° C.

In some embodiments, a heavy chain-only antibody is multi-specific. Insome embodiments, a heavy chain-only antibody is bispecific. In someembodiments, a heavy chain-only antibody binds to IL2RB and IL2RG. Insome embodiments, a heavy chain-only antibody functions as an IL2receptor beta/gamma agonist.

In some embodiments, a heavy chain-only antibody has an affinity forIL2R with a Kd of from about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., fromabout 10⁻¹⁰ M to around about 10⁻⁶ M; from about 10⁻⁹ M to around about10⁻⁶ M; from about 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M toaround about 10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; fromabout 10⁻⁹ M to around about 10⁻⁸ M; from about 10⁻¹¹ M to around about10⁻⁹ M; from about 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RG with a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M and anaffinity for IL2RG with a Kd of from about 10⁻⁹ M to around about2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include heavy chain-only antibodies that bindto IL2RG, comprising a heavy chain variable region comprising: (a) aCDR1 sequence having two or fewer (e.g., 0, 1, or 2) substitutions inany one of SEQ ID NOs: 15-16; and/or (b) a CDR2 sequence having two orfewer (e.g., 0, 1, or 2) substitutions in any one of SEQ ID NOs: 17-19;and/or (c) a CDR3 sequence having two or fewer (e.g., 0, 1, or 2)substitutions in any one of SEQ ID NOs: 20-21.

In some embodiments, a heavy chain-only antibody comprises: (a) a CDR1sequence comprising any one of SEQ ID NOs: 15-16; and/or (b) a CDR2sequence comprising any one of SEQ ID NOs: 17-19; and/or (c) a CDR3sequence comprising any one of SEQ ID NOs: 20-21.

In some embodiments, a heavy chain-only antibody comprises: (a) a CDR1sequence comprising any one of SEQ ID NOs: 15-16; and (b) a CDR2sequence comprising any one of SEQ ID NOs: 17-19; and (c) a CDR3sequence comprising any one of SEQ ID NOs: 20-21.

In some embodiments, a heavy chain-only antibody comprises:

-   -   (a) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID        NO: 17, and a CDR3 sequence of SEQ ID NO: 20, or    -   (b) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID        NO: 18, and a CDR3 sequence of SEQ ID NO: 20; or    -   (c) a CDR1 sequence of SEQ ID NO: 16, a CDR2 sequence of SEQ ID        NO: 18, and a CDR3 sequence of SEQ ID NO: 20; or    -   (d) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID        NO: 19, and a CDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, a heavy chain-only antibody comprises a heavy chainvariable region comprising a sequence having six or fewer (e.g., five orfewer, four or fewer, three or fewer, two or fewer; six, five, four,three, two, one, zero) substitutions in any one of SEQ ID NOs: 22-25. Insome embodiments, a heavy chain-only antibody comprises a heavy chainvariable region having at least 95% (e.g., at least 96%, at least 97%,at least 98%, at least 99%) sequence identity to any one of SEQ ID NOs:22-25. In some embodiments, a heavy chain-only antibody comprises aheavy chain variable region sequence selected from the group consistingof SEQ ID NOs: 22-25.

In some embodiments, a heavy-chain only antibody comprises a Fc region.In some embodiments, the heavy-chain only antibody comprises a variantFc region. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a heavy chain-only antibody further comprises aheavy chain constant region sequence in the absence of a CH1 sequence.In some embodiments, the heavy chain-only antibody comprises a heavychain constant region comprising a hinge region, a CH2 domain, and a CH3domain. In some embodiments, the hinge region comprises a wild typehuman IgG4 hinge region sequence (SEQ ID NO: 54).

In some embodiments, the hinge region comprises a variant human IgG4hinge region sequence comprising an S228P mutation (SEQ ID NO: 55). Insome embodiments, the CH2 domain comprises a wild type human IgG4 CH2domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domaincomprises a variant human IgG4 CH2 domain comprising an F234A mutation,an L235A mutation, or both an F234A mutation and an L235A mutation. Insome embodiments, the CH3 domain comprises a wild type human IgG4 CH3domain sequence (SEQ ID NO: 58).In some embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a heavy chain-only antibody has a Tm of from about55° C. to about 65° C. In some embodiments, a heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, aheavy chain-only antibody has a Tm of from about 55° C. to about 65° C.and a Tagg of from about 55° C. to about 65° C.

In some embodiments, a heavy chain-only antibody is multi-specific. Insome embodiments, a heavy chain-only antibody is bispecific. In someembodiments, a heavy chain-only antibody binds to IL2RB and IL2RG. Insome embodiments, a heavy chain-only antibody functions as an IL2receptor beta/gamma agonist.

In some embodiments, a heavy chain-only antibody has an affinity forIL2R with a Kd of from about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., fromabout 10⁻¹⁰ M to around about 10⁻⁶ M; from about 10⁻⁹ M to around about10⁻⁶ M; from about 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M toaround about 10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; fromabout 10⁻⁹ M to around about 10⁻⁸ M; from about 10⁻¹¹ M to around about10⁻⁹ M; from about 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RG with a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M and anaffinity for IL2RG with a Kd of from about 10⁻⁹ M to around about2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include heavy chain-only antibodies that bindto IL2RG, comprising a heavy chain variable region comprising:

-   -   (a) a CDR1 sequence comprising the formula:        -   G F X1 X2 X3 X4 Y Y (SEQ ID NO: 32)            where X1 is T or I; X2 is F or V; X3 is S, N, or G; and X4            is D or N;    -   (b) a CDR2 sequence comprising the formula:        -   I S X5 S G X6 X7 I (SEQ ID NO: 33)            where X5 is S or N; X6 is D, S, G, or N; and X7 is T or I;            and    -   (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID        NO: 20).

In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in aVH human framework.

In some embodiments, a heavy chain-only antibody is multi-specific. Insome embodiments, a heavy chain-only antibody is bispecific. In someembodiments, a heavy chain-only antibody binds to IL2RB and IL2RG.

In some embodiments, a heavy chain-only antibody comprises a Fc region.In some embodiments, a heavy chain-only antibody further comprises aheavy chain constant region comprising a hinge region, a CH2 domain, anda CH3 domain. In some embodiments, the heavy chain constant region doesnot contain a CH1 sequence. In some embodiments, the hinge regioncomprises a wild type human IgG4 hinge region sequence (SEQ ID NO: 54).In some embodiments, the hinge region comprises a variant human IgG4hinge region sequence comprising an S228P mutation (SEQ ID NO: 55). Insome embodiments, the CH2 domain comprises a wild type human IgG4 CH2domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domaincomprises a variant human IgG4 CH2 domain comprising an F234A mutation,an L235A mutation, or both an F234A mutation and an L235A mutation. Insome embodiments, the CH3 domain comprises a wild type human IgG4 CH3domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domaincomprises a variant human IgG4 CH3 domain sequence comprising a T366Wmutation. In some embodiments, the CH3 domain comprises a variant humanIgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and aY407V mutation.

In some embodiments, a heavy chain-only antibody has a Tm of from about55° C. to about 65° C. In some embodiments, a heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, aheavy chain-only antibody has a Tm of from about 55° C. to about 65° C.and a Tagg of from about 55° C. to about 65° C.

In some embodiments, a heavy chain-only antibody functions as an IL2receptor beta/gamma agonist.

In some embodiments, a heavy chain-only antibody has an affinity forIL2R with a Kd of from about 10⁻¹¹ M to around about 10⁻⁶ M (e.g., fromabout 10⁻¹⁰ M to around about 10⁻⁶ M; from about 10⁻⁹ M to around about10⁻⁶ M; from about 10⁻⁸ M to around about 10⁻⁶ M; from about 10⁻¹¹ M toaround about 10⁻⁸ M; from about 10⁻¹⁰ M to around about 10⁻⁸ M; fromabout 10⁻⁹ M to around about 10⁻⁸ M; from about 10⁻¹¹ M to around about10⁻⁹ M; from about 10⁻¹⁰ M to around about 10⁻⁹ M).

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RG with a Kd of from about 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, a heavy chain-only antibody has an affinity forIL2RB with a Kd of from about 10⁻⁸ M to around about 2.5×10⁻⁷ M and anaffinity for IL2RG with a Kd of from about 10⁻⁹ M to around about2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include a bispecific agonistic anti-IL2R heavychain-only antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of SEQ ID NO: 4; and aCDR3 sequence of SEQ ID NO: 7; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 17; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 11, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 22. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 11, and thesecond heavy chain variable region comprises SEQ ID NO: 22.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a Fc region. In some embodiments, a bispecificagonistic anti-IL2R heavy chain-only antibody comprises a variant Fcregion. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody further comprises a heavy chain constant region sequence in theabsence of a CH1 sequence. In some embodiments, the bispecific agonisticanti-IL2R heavy chain-only antibody comprises a heavy chain constantregion comprising a hinge region, a CH2 domain, and a CH3 domain. Insome embodiments, the hinge region comprises a wild type human IgG4hinge region sequence (SEQ ID NO: 54). In some embodiments, the hingeregion comprises a variant human IgG4 hinge region sequence comprisingan S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domaincomprises a wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). Insome embodiments, the CH2 domain comprises a variant human IgG4 CH2domain comprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a first polypeptide comprising SEQ ID NO: 53 and asecond polypeptide comprising SEQ ID NO: 61.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has a Tm of from about 55° C. to about 65° C. In someembodiments, a bispecific agonistic anti-IL2R heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, abispecific agonistic anti-IL2R heavy chain-only antibody has a Tm offrom about 55° C. to about 65° C. and a Tagg of from about 55° C. toabout 65° C.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2R with a Kd of from about 10⁻¹¹ M toaround about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M to around about 10⁻⁶ M;from about 10⁻⁹ M to around about 10⁻⁶ M; from about 10⁻⁸ M to aroundabout 10⁻⁶ M; from about 10⁻¹¹ M to around about 10⁻⁸ M; from about10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M to around about 10⁻⁸M; from about 10⁻¹¹ M to around about 10⁻⁹ M; from about 10⁻¹⁰ M toaround about 10⁻⁹ M).

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RG with a Kd of from about 10⁻⁹ M toaround about 2.5×10⁻⁸ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M and an affinity for IL2RG with a Kd of fromabout 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include a bispecific agonistic anti-IL2R heavychain-only antibody, comprising:

-   -   a first heavy chain variable region that binds to IL2RB,        comprising: a CDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of        SEQ ID NO: 4; and a CDR3 sequence of SEQ ID NO: 8; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 18; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 12, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 12, and thesecond heavy chain variable region comprises SEQ ID NO: 23.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a Fc region. In some embodiments, a bispecificagonistic anti-IL2R heavy chain-only antibody comprises a variant Fcregion. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody further comprises a heavy chain constant region sequence in theabsence of a CH1 sequence. In some embodiments, the bispecific agonisticanti-IL2R heavy chain-only antibody comprises a heavy chain constantregion comprising a hinge region, a CH2 domain, and a CH3 domain. Insome embodiments, the hinge region comprises a wild type human IgG4hinge region sequence (SEQ ID NO: 54). In some embodiments, the hingeregion comprises a variant human IgG4 hinge region sequence comprisingan S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domaincomprises a wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). Insome embodiments, the CH2 domain comprises a variant human IgG4 CH2domain comprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a first polypeptide comprising SEQ ID NO: 62 and asecond polypeptide comprising SEQ ID NO: 63.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has a Tm of from about 55° C. to about 65° C. In someembodiments, a bispecific agonistic anti-IL2R heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, abispecific agonistic anti-IL2R heavy chain-only antibody has a Tm offrom about 55° C. to about 65° C. and a Tagg of from about 55° C. toabout 65° C.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2R with a Kd of from about 10⁻¹¹ M toaround about 10⁻⁶ M (e.g., from about 10⁻¹⁰ to around about 10⁻⁶ M; fromabout 10⁻⁹ M to around about 10⁻⁶ M; from about 10⁻⁸ M to around about10⁻⁶ M; from about 10⁻¹¹ M to around about 10⁻⁸ M; from about 10⁻¹⁰ M toaround about 10⁻⁸ M; from about 10⁻⁹ M to around about 10⁻⁸ M; fromabout 10⁻¹¹ M to around about 10⁻⁹ M; from about 10⁻¹⁰ M to around about10⁻⁹ M).

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RG with a Kd of from about 10⁻⁹ M toaround about 2.5×10⁻⁸ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M and an affinity for IL2RG with a Kd of fromabout 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include a bispecific agonistic anti-IL2R heavychain-only antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 2; a CDR2 sequence of SEQ ID NO: 5; and aCDR3 sequence of SEQ ID NO: 9; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 18; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 13; and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 13, and thesecond heavy chain variable region comprises SEQ ID NO: 23.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a Fc region. In some embodiments, a bispecificagonistic anti-IL2R heavy chain-only antibody comprises a variant Fcregion. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody further comprises a heavy chain constant region sequence in theabsence of a CH1 sequence. In some embodiments, the bispecific agonisticanti-IL2R heavy chain-only antibody comprises a heavy chain constantregion comprising a hinge region, a CH2 domain, and a CH3 domain. Insome embodiments, the hinge region comprises a wild type human IgG4hinge region sequence (SEQ ID NO: 54). In some embodiments, the hingeregion comprises a variant human IgG4 hinge region sequence comprisingan S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domaincomprises a wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). Insome embodiments, the CH2 domain comprises a variant human IgG4 CH2domain comprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a first polypeptide comprising SEQ ID NO: 64 and asecond polypeptide comprising SEQ ID NO: 65.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has a Tm of from about 55° C. to about 65° C. In someembodiments, a bispecific agonistic anti-IL2R heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, abispecific agonistic anti-IL2R heavy chain-only antibody has a Tm offrom about 55° C. to about 65° C. and a Tagg of from about 55° C. toabout 65° C.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2R with a Kd of from about 10⁻¹¹ M toaround about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M to around about 10⁻⁶ M;from about 10⁻⁹ M to around about 10⁻⁶ M; from about 10⁻⁸ M to aroundabout 10⁻⁶ M; from about 10⁻¹¹ M to around about 10⁻⁸ M; from about10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M to around about 10⁻⁸M; from about 10⁻¹¹ M to around about 10⁻⁹ M; from about 10⁻¹⁰ M toaround about 10⁻⁹ M).

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RG with a Kd of from about 10⁻⁹ M toaround about 2.5×10⁻⁸ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M and an affinity for IL2RG with a Kd of fromabout 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include a bispecific agonistic anti-IL2R heavychain-only antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 3; a CDR2 sequence of SEQ ID NO: 6; and aCDR3 sequence of SEQ ID NO: 10; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 17; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 14, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 22. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 14, and thesecond heavy chain variable region comprises SEQ ID NO: 22.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a Fc region. In some embodiments, a bispecificagonistic anti-IL2R heavy chain-only antibody comprises a variant Fcregion. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody further comprises a heavy chain constant region sequence in theabsence of a CH1 sequence. In some embodiments, the bispecific agonisticanti-IL2R heavy chain-only antibody comprises a heavy chain constantregion comprising a hinge region, a CH2 domain, and a CH3 domain. Insome embodiments, the hinge region comprises a wild type human IgG4hinge region sequence (SEQ ID NO: 54). In some embodiments, the hingeregion comprises a variant human IgG4 hinge region sequence comprisingan S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domaincomprises a wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). Insome embodiments, the CH2 domain comprises a variant human IgG4 CH2domain comprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a first polypeptide comprising SEQ ID NO: 66 and asecond polypeptide comprising SEQ ID NO: 67.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has a Tm of from about 55° C. to about 65° C. In someembodiments, a bispecific agonistic anti-IL2R heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, abispecific agonistic anti-IL2R heavy chain-only antibody has a Tm offrom about 55° C. to about 65° C. and a Tagg of from about 55° C. toabout 65° C.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2R with a Kd of from about 10⁻¹¹ M toaround about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M to around about 10⁻⁶ M;from about 10⁻⁹ M to around about 10⁻⁶ M; from about 10⁻⁸ M to aroundabout 10⁻⁶ M; from about 10⁻¹¹ M to around about 10⁻⁸ M; from about10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M to around about 10⁻⁸M; from about 10⁻¹¹ M to around about 10⁻⁹ M; from about 10⁻¹⁰ M toaround about 10⁻⁹ M).

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RG with a Kd of from about 10⁻⁹ M toaround about 2.5×10⁻⁸ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M and an affinity for IL2RG with a Kd of fromabout 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include a bispecific agonistic anti-IL2R heavychain-only antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of SEQ ID NO: 4; and aCDR3 sequence of SEQ ID NO: 8; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 16; a CDR2 sequence of SEQ ID NO: 18; and aCDR3 sequence of SEQ ID NO: 20.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 12; and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 24. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 12, and thesecond heavy chain variable region comprises SEQ ID NO: 24.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a Fc region. In some embodiments, a bispecificagonistic anti-IL2R heavy chain-only antibody comprises a variant Fcregion. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody further comprises a heavy chain constant region sequence in theabsence of a CH1 sequence. In some embodiments, the bispecific agonisticanti-IL2R heavy chain-only antibody comprises a heavy chain constantregion comprising a hinge region, a CH2 domain, and a CH3 domain. Insome embodiments, the hinge region comprises a wild type human IgG4hinge region sequence (SEQ ID NO: 54). In some embodiments, the hingeregion comprises a variant human IgG4 hinge region sequence comprisingan S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domaincomprises a wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). Insome embodiments, the CH2 domain comprises a variant human IgG4 CH2domain comprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a first polypeptide comprising SEQ ID NO: 34 and asecond polypeptide comprising SEQ ID NO: 35.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has a Tm of from about 55° C. to about 65° C. In someembodiments, a bispecific agonistic anti-IL2R heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, abispecific agonistic anti-IL2R heavy chain-only antibody has a Tm offrom about 55° C. to about 65° C. and a Tagg of from about 55° C. toabout 65° C.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2R with a Kd of from about 10⁻¹¹ M toaround about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M to around about 10⁻⁶ M;from about 10⁻⁹ M to around about 10⁻⁶ M; from about 10⁻⁸ M to aroundabout 10⁻⁶ M; from about 10⁻¹¹ M to around about 10⁻⁸ M; from about10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M to around about 10⁻⁸M; from about 10⁻¹¹ M to around about 10⁻⁹ M; from about 10⁻¹⁰ M toaround about 10⁻⁹ M).

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RG with a Kd of from about 10⁻⁹ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M and an affinity for IL2RG with a Kd of fromabout 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include a bispecific agonistic anti-IL2R heavychain-only antibody, comprising:

a first heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence of SEQ ID NO: 1; a CDR2 sequence of SEQ ID NO: 4; and aCDR3 sequence of SEQ ID NO: 8; and

a second heavy chain variable region that binds to IL2RG, comprising: aCDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 19; and aCDR3 sequence of SEQ ID NO: 21.

In some embodiments, the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the second heavychain variable region are present in a VH human framework. In someembodiments, the CDR1, CDR2, and CDR3 sequences in the first and secondheavy chain variable regions are present in VH human frameworks.

In some embodiments, the first heavy chain variable region has at least95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%)sequence identity to SEQ ID NO: 12, and the second heavy chain variableregion has at least 95% (e.g., at least 96%, at least 97%, at least 98%,at least 99%) sequence identity to SEQ ID NO: 25. In some embodiments,the first heavy chain variable region comprises SEQ ID NO: 12, and thesecond heavy chain variable region comprises SEQ ID NO: 25.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a Fc region. In some embodiments, a bispecificagonistic anti-IL2R heavy chain-only antibody comprises a variant Fcregion. In some embodiments, the variant Fc region possesses at leastabout 80% (e.g., at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%) homology with anative-sequence Fc region.

In some embodiments, the variant Fc region comprises heterodimerizingalterations. In some embodiments, the heterodimerizing alterationscomprise knob and holes substitutions (e.g., in a variant IgG1 Fcregion, 1) Y407T in one chain and T366Y in the other; 2) Y407A in onechain and T366W in the other; 3) F405A in one chain and T394W in theother; 4) F405W in one chain and T394S in the other; 5) Y407T in onechain and T366Y in the other; 6) T366Y and F405A in one chain and T394Wand Y407T in the other; 7) T366W and F405W in one chain and T394S andY407A in the other; 8) F405W and Y407A in one chain and T366W and T394Sin the other; or 9) T366W in one polypeptide of the Fc and T366S, L368A,and Y407V in the other). In some embodiments, the heterodimerizingalterations comprise substitutions that create new disulfide bridges(e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chainand S354C in the other; 2) Y349C in one Fc polypeptide chain and E356Cin the other; 3) Y349C in one Fc polypeptide chain and E357C in theother; 4) L351C in one Fc polypeptide chain and S354C in the other; 5)T394C in one Fc polypeptide chain and E397C in the other; or 6) D399C inone Fc polypeptide chain and K392C in the other). In some embodiments,the heterodimerizing alterations comprise charge pair substitutions(e.g., 1) K409E in one chain plus D399K in the other; 2) K409E in onechain plus D399R in the other; 3) K409D in one chain plus D399K in theother; 4) K409D in one chain plus D399R in the other; 5) K392E in onechain plus D399R in the other; 6) K392E in one chain plus D399K in theother; 7) K392D in one chain plus D399R in the other; 8) K392D in onechain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; or 17) K409D and K439D on one chain plus D399K and E356K on theother).

In some embodiments, the Fc region is a silenced Fc region. In someembodiments, the silenced Fc region comprises substitution of one ormore (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297,327 and 329 according to EU numbering. In some embodiments, the silencedFc region comprises a substitution that alters glycosylation. In someembodiments, the silenced Fc region comprises an effector-less mutation(e.g., an N297A, an N297G, a DANA mutation (D265A+N297A), or a DANGmutation (D265A+N297G) in the CH2 region). In some embodiments, thesilenced Fc region comprises K322A and L234A/L235A mutations.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody further comprises a heavy chain constant region sequence in theabsence of a CH1 sequence. In some embodiments, the bispecific agonisticanti-IL2R heavy chain-only antibody comprises a heavy chain constantregion comprising a hinge region, a CH2 domain, and a CH3 domain. Insome embodiments, the hinge region comprises a wild type human IgG4hinge region sequence (SEQ ID NO: 54). In some embodiments, the hingeregion comprises a variant human IgG4 hinge region sequence comprisingan S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domaincomprises a wild type human IgG4 CH2 domain sequence (SEQ ID NO: 56). Insome embodiments, the CH2 domain comprises a variant human IgG4 CH2domain comprising an F234A mutation, an L235A mutation, or both an F234Amutation and an L235A mutation. In some embodiments, the CH3 domaincomprises a wild type human IgG4 CH3 domain sequence (SEQ ID NO: 58). Insome embodiments, the CH3 domain comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation. In some embodiments, theCH3 domain comprises a variant human IgG4 CH3 domain sequence comprisinga T366S, an L368A mutation, and a Y407V mutation.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody comprises a first polypeptide comprising SEQ ID NO: 36 and asecond polypeptide comprising SEQ ID NO: 37.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has a Tm of from about 55° C. to about 65° C. In someembodiments, a bispecific agonistic anti-IL2R heavy chain-only antibodyhas a Tagg of from about 55° C. to about 65° C. In some embodiments, abispecific agonistic anti-IL2R heavy chain-only antibody has a Tm offrom about 55° C. to about 65° C. and a Tagg of from about 55° C. toabout 65° C.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2R with a Kd of from about 10⁻¹¹ M toaround about 10⁻⁶ M (e.g., from about 10⁻¹⁰ M to around about 10⁻⁶ M;from about 10⁻⁹ M to around about 10⁻⁶ M; from about 10⁻⁸ M to aroundabout 10⁻⁶ M; from about 10⁻¹¹ M to around about 10⁻⁸ M; from about10⁻¹⁰ M to around about 10⁻⁸ M; from about 10⁻⁹ M to around about 10⁻⁸M; from about 10⁻¹¹ M to around about 10⁻⁹ M; from about 10⁻¹⁰ M toaround about 10⁻⁹ M).

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RG with a Kd of from about 10⁻⁹ M toaround about 2.5×10⁻⁸ M.

In some embodiments, a bispecific agonistic anti-IL2R heavy chain-onlyantibody has an affinity for IL2RB with a Kd of from about 10⁻⁸ M toaround about 2.5×10⁻⁷ M and an affinity for IL2RG with a Kd of fromabout 10⁻⁹ M to around about 2.5×10⁻⁸ M.

In some embodiments, the Kd is measured using a ForteBio Octet Qk384instrument in kinetics mode. In some embodiments, the Kd is measuredusing a ForteBio Octet Qk384 instrument comprising an anti-human Fccapture (AHC, 18-5005) sensor in kinetics mode. In some embodiments, theKd is measured according to a method described in the Examples herein.

Aspects of the disclosure include pharmaceutical compositions comprisingan antibody (e.g., a heavy chain-only antibody; a bispecific agonisticanti-IL2R heavy chain-only antibody) as described herein.

Aspects of the disclosure include polynucleotides encoding an antibody(e.g., a heavy chain-only antibody; a bispecific agonistic anti-IL2Rheavy chain-only antibody) as described herein.

Aspects of the disclosure include vectors comprising a polynucleotide asdescribed herein.

Aspects of the disclosure include cells comprising the vectors asdescribed herein.

Aspects of the disclosure include methods of producing an antibody(e.g., a heavy chain-only antibody; a bispecific agonistic anti-IL2Rheavy chain-only antibody) as described herein, the methods comprisinggrowing a cell as described herein under conditions permissive forexpression of the antibody, and isolating the antibody from the celland/or a cell culture medium in which the cell is grown.

Aspects of the disclosure include methods of making an antibody asdescribed herein, the methods comprising immunizing a UniRat™ animalwith IL2R and identifying IL2R-binding heavy chain sequences.

Aspects of the disclosure include kits for treating a disease ordisorder in an individual in need, comprising an antibody (e.g., a heavychain-only antibody; a bispecific agonistic anti-IL2R heavy chain-onlyantibody) as described herein, or a pharmaceutical composition asdescribed herein, and instructions for use.

In some embodiments, a kit further comprises at least one additionalreagent. In some embodiments, the at least one additional reagentcomprises a chemotherapeutic drug.

Aspects of the disclosure include methods of treatment, comprisingadministering to an individual in need an effective dose of an antibody(e.g., a heavy chain-only antibody; a bispecific agonistic anti-IL2Rheavy chain-only antibody) as described herein, or a pharmaceuticalcomposition as described herein.

Aspects of the disclosure include use of an antibody (e.g., a heavychain-only antibody; a bispecific agonistic anti-IL2R heavy chain-onlyantibody) as described herein in the preparation of a medicament for thetreatment of a disease or disorder in an individual in need. In someembodiments, the disease or disorder is a cancer. In some embodiments,the cancer is an advanced or metastatic cancer. In some embodiments, thecancer is a liquid cancer, such as, e.g., multiple myeloma or acutemyeloid leukemia. In some embodiments, the cancer is a solid tumorcancer. In some embodiments, the solid tumor cancer is selected from thegroup consisting of renal cell carcinoma, melanoma, urothelial cancer,triple negative breast cancer, non-small cell lung cancer (NSCLC),colorectal cancer, sarcoma, squamous cell carcinoma of the head andneck, and metastatic castration-resistant prostate cancer.

Aspects of the disclosure include an antibody (e.g., a heavy chain-onlyantibody; a bispecific agonistic anti-IL2R heavy chain-only antibody) asdescribed herein, or a pharmaceutical composition as described herein,for use in therapy in an individual in need.

Aspects of the disclosure include an antibody (e.g., a heavy chain-onlyantibody; a bispecific agonistic anti-IL2R heavy chain-only antibody) asdescribed herein, or a pharmaceutical composition as described herein,for use in the treatment of a disease or disorder in an individual inneed. In some embodiments, the disease or disorder is a cancer. In someembodiments, the cancer is an advanced or metastatic cancer. In someembodiments, the cancer is a liquid cancer, such as, e.g., multiplemyeloma or acute myeloid leukemia. In some embodiments, the cancer is asolid tumor cancer. In some embodiments, the solid tumor cancer isselected from the group consisting of renal cell carcinoma, melanoma,urothelial cancer, triple negative breast cancer, non-small cell lungcancer (NSCLC), colorectal cancer, sarcoma, squamous cell carcinoma ofthe head and neck, and metastatic castration-resistant prostate cancer.

Aspects of the disclosure include methods for the treatment of a cancer,comprising administering to a subject with said cancer an antibody(e.g., a heavy chain-only antibody; a bispecific agonistic anti-IL2Rheavy chain-only antibody) as described herein, or a pharmaceuticalcomposition as described herein. In some embodiments, the cancer is anadvanced or metastatic cancer. In some embodiments, the cancer is aliquid cancer, such as, e.g., multiple myeloma or acute myeloidleukemia. In some embodiments, the cancer is a solid tumor cancer. Insome embodiments, the solid tumor cancer is selected from the groupconsisting of renal cell carcinoma, melanoma, urothelial cancer, triplenegative breast cancer, non-small cell lung cancer (NSCLC), colorectalcancer, sarcoma, squamous cell carcinoma of the head and neck, andmetastatic castration-resistant prostate cancer.

Aspects of the disclosure include methods for stimulating IL2R signalingin an immune cell, the methods comprising contacting the immune cellwith an antibody (e.g., a heavy chain-only antibody; a bispecificagonistic anti-IL2R heavy chain-only antibody) as described herein, or apharmaceutical composition as described herein.

Aspects of the disclosure include methods for stimulating an IL2RB/IL2RGdimeric receptor complex on an immune cell, the method comprisingcontacting the immune cell with an antibody (e.g., a heavy chain-onlyantibody; a bispecific agonistic anti-IL2R heavy chain-only antibody) asdescribed herein, or a pharmaceutical composition as described herein.

In some embodiments, the immune cell is selected from the groupconsisting of a CD4+ T-cell, a CD8+ T-cell, and a Natural Killer (NK)cell.

These and further aspects will be further explained in the rest of thedisclosure, including in the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table summarizing the binding kinetics of the listedbispecific antibody constructs with respect to human and cynomolgusIL2RB and IL2RG.

FIG. 2A, FIG. 2B and FIG. 2C are heatmap tables depicting fold-inductionof phosphorylated STAT5 (pSTAT5) in CD8⁺ T-cells from human PBMCstreated with: anti-IL2Rβ/γ bispecific UniAbs™ (FIG. 2A); anti-IL2Rβ andanti-IL2Rγ monospecific UniAbs™ in a 1:1 mixture or as single agents(FIG. 2B); or IL-2 as a control (FIG. 2C) at 50 nM for 1 hour. pSTAT5levels were determined by flow cytometry and reported as geometric meanfluorescent intensity (gMFI) over the gMFI of unstimulated cells.

FIG. 3A, FIG. 3B, and FIG. 3C are graphs showing cell binding of theindicated cell type as a function of concentration for the depictedbispecific antibody constructs. Cell binding was determined by flowcytometry and reported as geometric mean fluorescent intensity (gMFI)over the gMFI of cells stained only with secondary detection antibody.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E are graphs showing STAT5phosphorylation dose curves in human and cyno PBMCs as a function ofconcentration for the depicted bispecific antibody constructs andcontrol molecules (IL-2 and IL-2 variant). pSTAT5 levels were determinedby flow cytometry and reported as a percentage of the indicated celltype.

FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D are graphs showing proliferation(Ki67 dose curves) in the indicated human cells as a function ofconcentration for the depicted bispecific antibody constructs andcontrol molecules (IL-2 and IL-2 variant). Ki67 levels were determinedby flow cytometry and were reported as a percentage of the indicatedcell type.

FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D are graphs showing cytokinesecretion in human whole blood as a function of concentration for thedepicted bispecific antibody constructs and control molecules (IL-2).

FIG. 7A and FIG. 7B provide cellular internalization data for theindicated bispecific antibody constructs. FIG. 7A depictsinternalization of the indicated anti-IL2Rβ/γ UniAbs by CD8+ T-cellsfrom human PBMCs, as a function of time. FIG. 7B provides depicts thisdata in tabular format. Surface levels of UniAb were detected by flowcytometry and reported relative to cells which had not been allowed tointernalize. The observed half-lives ranged from 0.27 hours to 0.81hours. As observed here, internalization was potentially partiallydependent on the specific anti-IL2RG arm of the bispecific antibody, asmolecules comprising the IL2RG_F16B binding sequence internalizedfaster, and to a greater degree, than molecules containing differentanti-IL2RG binding sequences.

FIG. 8A and FIG. 8B provide mouse model PK data in graphical (FIG. 8A)and tabular (FIG. 8B) formats. BALB/c mice (n=3 per group per timepoint) were administered 1 mg/kg of the indicated anti-IL2Rβ/γ UniAbs bytail-vein injection. Serum was collected at 6 time points over two weeksand tested together by ELISA for human IgG4. Results are shown as afunction of time (FIG. 8A) or in tabular format (FIG. 8B).

FIG. 9 is a table summarizing several properties of the indicatedbispecific antibody constructs. All constructs were expressed in anExpiCHO expression system and were 2-step purified. Stability wasdetermined based on percent aggregation by SE-HPLC after thermal stress.Tm and Tagg were measured using the UNcle platform. For SE-HPLCexperiments, 20 μg of protein was run on TSK gel G3000 5 μm column.

FIG. 10A, FIG. 10B and FIG. 10C provide summary data from a mouse modelof GVHD. Irradiated NSG mice (5 per treatment group) were engrafted with20 million human PBMCs each. Animals were then treated with eithervehicle only (100 μL), 22 μg rhIL-2 daily, or one of the two indicatedbispecific antibody constructs at 1 mg/kg in 100 μL twice a week untilsacrifice (20% body weight loss). FIG. 10A provides an overview of themouse model of GVHD and subsequent dosing scheme. FIG. 10B shows animalbody weights as a function of time for the indicated experimentalgroups. FIG. 10C depicts an analysis of cells from spleens of the micein the study, harvested after day 5 of treatment. Proliferation of CD8+T-cells and CD4+ T-cells was compared between the 4 treatment groups bymeasuring CSFE staining in the different lymphocyte populations. The twotested bispecific antibody constructs (IL2RB_F09C**IL2RG_F16A (BsAb-1)and IL2RB_F09G**IL2RG_F16B (BsAb-2)) both showed significantly moreproliferating CD8+ T-cells compared to rhIL-2 and the vehicle control.CD4+ T-cells were expanded to a lesser extent; however, a significantincrease in proliferating CD4+ T-cells was seen inIL2RB_F09G**IL2RG_F16B (BsAb-2)-treated mice compared to the vehiclecontrol (FIG. 10C). The data demonstrate that cytokine receptor agonistspromote immune effector activation and proliferation in vivo andaccelerate GVHD in huPBMC-engrafted NSG mice at a rate similar tocytokine controls.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11G,FIG. 11H, FIG. 11I, FIG. 11J and FIG. 11K are graphs summarizing in vivopharmacodynamic (PD) data from a non-GLP cynomolgus monkey study. FIGS.11A-11E depict the percentages of the indicated cell types as a functionof time post dose. FIGS. 11F-11J depict the concentration of theindicated cell types per μL of blood (×10⁵) as a function of time postdose. FIG. 11K shows the ratio of CD8+ T-cells to CD4+ T-cells as afunction of time post dose.

FIG. 12A is a graph showing serum concentration as a function of time(days) for the indicated bispecific antibody.

FIG. 12B is a table showing molecule, dose and half life (t_(1/2))information.

FIG. 13 is a graph showing body weight (%) as a function of time (studyday) for animals in an accelerated GVHD model.

FIG. 14 is a graph showing probability of survival as a function of time(study day) for animals in an accelerated GVHD model.

FIG. 15A, FIG. 15B and FIG. 15C are graphs showing cell proliferation ofthe indicated cell type, measured at 5 days post-treatment, andseparated into treatment groups. FIG. 15A shows results for CD8+T-cells, FIG. 15B shows results for CD4+ T-cells, and FIG. 15C showsresults for NK-cells.

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G,FIG. 16H, FIG. 16I, FIG. 16J, FIG. 16K and FIG. 16L are graphs showingcell proliferation and absolute cell concentration for the indicatedcell types under the indicated dosing conditions, as a function of time.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: ALaboratory Manual: Portable Protocol No. I, Cold Spring HarborLaboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory; (1988).

Unless indicated otherwise, antibody residues herein are numberedaccording to the Kabat numbering system (e.g., Kabat et al., Sequencesof Immunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present disclosure.However, it will be apparent to one of skill in the art that the presentdisclosure may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the disclosure.

All references cited throughout the disclosure, including patentapplications and publications, are incorporated by reference herein intheir entirety. Where there is any discrepancy in definition between thecited references and the definitions provided herein, the definitionsprovided herein control.

Definitions:

In some embodiments, “about,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g., within the 95% confidence interval for the mean)or ±10% of the indicated value, whichever is greater. In someembodiments, numeric ranges are inclusive of the numbers defining therange.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure.

By “comprising”, it is meant that the recited elements are required inthe composition/method/kit, but other elements may be included to formthe composition/method/kit etc. within the scope of the claim orembodiment.

By “consisting essentially of”, it is meant a limitation of the scope ofcomposition or method described to the specified materials or steps thatdo not materially affect the basic and novel characteristic(s) of thesubject disclosure.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim or embodiment.

Antibody residues herein are numbered according to the Kabat numberingsystem and the EU numbering system. The Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately residues 1-113 of the heavy chain) (e.g., Kabat et al.,Sequences of Immunological Interest. 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The “EU numberingsystem” or “EU index” is generally used when referring to a residue inan immunoglobulin heavy chain constant region (e.g., the EU indexreported in Kabat et al., supra). The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody. Unless statedotherwise herein, references to residue numbers in the variable domainof antibodies mean residue numbering by the Kabat numbering system.Unless stated otherwise herein, references to residue numbers in theconstant domain of antibodies mean residue numbering by the EU numberingsystem.

Antibodies, also referred to as immunoglobulins, conventionally compriseat least one heavy chain and one light chain, where the amino terminaldomain of the heavy and light chains is variable in sequence, and henceis commonly referred to as a variable region domain, or a variable heavy(VH) or variable light (VL) domain. The two domains conventionallyassociate to form a specific binding region, although as will bediscussed here, specific binding can also be obtained with heavychain-only variable sequences, and a variety of non-naturalconfigurations of antibodies are known and used in the art.

A “functional” or “biologically active” antibody or antigen-bindingmolecule (including, e.g., heavy chain-only antibodies andmulti-specific (e.g., bispecific) antibodies, as well as three-chainantibody-like molecules (TCAs, described herein)) is one capable ofexerting one or more of its natural activities in structural,regulatory, biochemical, or biophysical events. For example, afunctional antibody or other binding molecule, e.g., a TCA, may have theability to specifically bind an antigen and the binding may in turnelicit or alter a cellular or molecular event such as signaltransduction or enzymatic activity. A functional antibody or otherbinding molecule, e.g., a TCA, may also block ligand activation of areceptor or act as an agonist or antagonist. The capability of anantibody or other binding molecule, e.g., a TCA, to exert one or more ofits natural activities depends on several factors, including properfolding and assembly of the polypeptide chains.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,monomers, dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), heavy chain-only antibodies, three chain antibodies, TCAs,single chain Fv (scFv), nanobodies, etc., and also includes antibodyfragments, so long as they exhibit the desired biological activity.Miller et al (2003) Jour. of Immunology 170:4854-4861. Antibodies may bemurine, human, humanized, chimeric, or derived from other species.

For example, the term “antibody” may reference a full-length heavychain, a full-length light chain, an intact immunoglobulin molecule, oran immunologically active portion of any of these polypeptides, i.e., apolypeptide that comprises an antigen binding site thatimmunospecifically binds an antigen of a target of interest or partthereof, such targets including, but not limited to, a cancer cell orcells that produce autoimmune antibodies associated with an autoimmunedisease. The immunoglobulin disclosed herein can be of any type (e.g.,IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule, including engineeredsubclasses with altered Fc portions that provide for reduced or enhancedeffector cell activity. Light chains of the subject antibodies can bekappa light chains (Vkappa) or lambda light chains (Vlambda). Theimmunoglobulins can be derived from any species. In one aspect, theimmunoglobulin is of largely human origin.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. Monoclonal antibodies in accordance with the present disclosurecan be made by the hybridoma method first described by Kohler et al.(1975) Nature 256:495, and can also be made via recombinant proteinproduction methods (see, e.g., U.S. Pat. No. 4,816,567), for example.

The term “variable”, as used in connection with antibodies, refers tothe fact that certain portions of the antibody variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed throughout thevariable domains of antibodies. It is concentrated in three segmentscalled hypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FRs). The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, MD. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region”, when used herein, refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR” (e.g., residues 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD.(1991)) and/or those residues from a “hypervariable loop” residues 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments,“CDR” means a complementarity-determining region of an antibody asdefined in Lefranc, M P et al., IMGT, the International ImMunoGeneTicsdatabase, Nucleic Acids Res., 27:209-212 (1999). “Framework Region” or“FR” residues are those variable domain residues other than thehypervariable region/CDR residues as herein defined.

Exemplary CDR designations are shown herein; however, one of skill inthe art will understand that a number of definitions of the CDRs arecommonly in use, including the Kabat definition (see Zhao et al. “Agermline knowledge based computational approach for determining antibodycomplementarity determining regions.” Mol Immunol. 2010; 47:694-700),which is based on sequence variability and is the most commonly used.The Chothia definition is based on the location of the structural loopregions (Chothia et al. “Conformations of immunoglobulin hypervariableregions.” Nature. 1989; 342:877-883). Alternative CDR definitions ofinterest include, without limitation, those disclosed by Honegger, “Yetanother numbering scheme for immunoglobulin variable domains: anautomatic modeling and analysis tool.” J Mol Biol. 2001; 309:657-670;Ofran et al. “Automated identification of complementarity determiningregions (CDRs) reveals peculiar characteristics of CDRs and B-cellepitopes.” J Immunol. 2008; 181:6230-6235; Almagro “Identification ofdifferences in the specificity-determining residues of antibodies thatrecognize antigens of different size: implications for the rationaldesign of antibody repertoires.” J Mol Recognit. 2004; 17:132-143; andPadlan et al. “Identification of specificity-determining residues inantibodies.” Faseb J. 1995; 9:133-139., each of which is hereinspecifically incorporated by reference.

The terms “heavy chain-only antibody” and “heavy chain antibody” areused interchangeably herein and refer, in the broadest sense, toantibodies, or one or more portions of an antibody, e.g., one or morearms of an antibody, lacking the light chain of a conventional antibody(i.e., a “heavy chain antibody” may consist of an isolated portion of anantibody or an antibody format other than a conventional antibody thatlacks a light chain). The terms specifically include, withoutlimitation, homodimeric antibodies comprising the VH antigen-bindingdomain and the CH2 and CH3 constant domains, in the absence of the CH1domain; functional (antigen-binding) variants of such antibodies,soluble VH variants, Ig-NAR comprising a homodimer of one variabledomain (V-NAR) and five C-like constant domains (C-NAR) and functionalfragments thereof; and soluble single domain antibodies (e.g.,UniDabs™). In one embodiment, a heavy chain-only antibody is composed ofa variable region antigen-binding domain composed of framework 1, CDR1,framework 2, CDR2, framework 3, CDR3, and framework 4. In anotherembodiment, a heavy chain-only antibody is composed of anantigen-binding domain, at least part of a hinge region, and CH2 and CH3domains. In another embodiment, a heavy chain-only antibody is composedof an antigen-binding domain, at least part of a hinge region, and a CH2domain. In a further embodiment, a heavy chain-only antibody is composedof an antigen-binding domain, at least part of a hinge region, and a CH3domain. Heavy chain-only antibodies in which the CH2 and/or CH3 domainis truncated are also included herein. In a further embodiment, a heavychain is composed of an antigen binding domain, and at least one CH(CH1, CH2, CH3, or CH4) domain but no hinge region. A heavy chain-onlyantibody can be in the form of a dimer, in which two heavy chains aredisulfide bonded or otherwise, covalently or non-covalently, attachedwith each other. The heavy chain-only antibody may belong to the IgGsubclass, but antibodies belonging to other subclasses, such as IgM,IgA, IgD and IgE subclass, are also included herein. In a particularembodiment, a heavy chain antibody may belong to the IgG1, IgG2, IgG3,or IgG4 subtype, e.g., the IgG1 or IgG4 subtype. In one embodiment, theheavy-chain antibody is of the IgG1 or IgG4 subtype, wherein one or moreof the CH domains is modified to alter an effector function of theantibody. In one embodiment, a heavy-chain antibody is of the IgG4subtype, wherein one or more of the CH domains is modified to alter aneffector function of the antibody. In one embodiment, the heavy-chainantibody is of the IgG1 subtype, wherein one or more of the CH domainsis modified to alter an effector function of the antibody. Modificationsof CH domains that alter effector function are further described herein.Non-limiting examples of heavy-chain antibodies are described, forexample, in WO2018/039180, the disclosure of which is incorporatedherein by reference in its entirety.

In some embodiments, the heavy chain-only antibodies described hereinare used as a binding (targeting) domain of a chimeric antigen receptor(CAR). The definition specifically includes human heavy chain-onlyantibodies produced by human immunoglobulin transgenic rats (e.g.,UniRat™), such as, e.g., UniAbs™. The variable regions (VH) of UniAbs™are called UniDabs™ and are versatile building blocks that can be linkedto Fc regions or serum albumin for the development of novel therapeuticswith multi-specificity, increased potency, and extended half-life. Sincethe homodimeric UniAbs™ lack a light chain and thus a VL domain, theantigen is recognized by one single domain, i.e., the variable domain ofthe heavy chain of a heavy-chain antibody (VH or VHH).

An “intact antibody chain” as used herein is one comprising afull-length variable region and a full length constant region (Fc). Anintact “conventional” antibody comprises an intact light chain and anintact heavy chain, as well as a light chain constant domain (CL) andheavy chain constant domains, CH1, hinge, CH2 and CH3 for secreted IgG.Other isotypes, such as IgM or IgA may have different CH domains. Theconstant domains may be native sequence constant domains (e.g., humannative sequence constant domains) or amino acid sequence variantsthereof. The intact antibody may have one or more “effector functions”which refer to those biological activities attributable to the Fcconstant region (a native sequence Fc region or amino acid sequencevariant Fc region) of an antibody. Examples of antibody effectorfunctions include C1q binding; complement dependent cytotoxicity; Fcreceptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; and down regulation of cell surface receptors. Constantregion variants include those that alter the effector profile, bindingto Fc receptors, and the like.

Depending on the amino acid sequence of the Fc (constant domain) oftheir heavy chains, antibodies and various antigen-binding proteins canbe provided as different classes. There are five major classes of heavychain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA, and IgA2. The Fc constant domains that correspond to thedifferent classes of antibodies may be referenced as α, δ, ε, γ, and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Ig forms include hinge-modifications or hingeless forms. Roux et al(1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem.267:7246-7256; US 2005/0048572; US 2004/0229310. The light chains ofantibodies from any vertebrate species can be assigned to one of twotypes, called κ (kappa) and λ (lambda), based on the amino acidsequences of their constant domains. Antibodies in accordance withembodiments of the disclosure can comprise kappa light chain sequencesor lambda light chain sequences.

A “functional Fc region” possesses an “effector function” of anative-sequence Fc region. Non-limiting examples of effector functionsinclude C1q binding; CDC; Fc-receptor binding; ADCC; ADCP;down-regulation of cell-surface receptors (e.g., B-cell receptor), etc.Such effector functions generally require the Fc region to interact witha receptor, e.g., the FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA;FcγRIIIB receptors, and the low affinity FcRn receptor; and can beassessed using various assays known in the art. A “dead” or “silenced”Fc is one that has been mutated to retain activity with respect to, forexample, prolonging serum half-life, but which does not activate a highaffinity Fc receptor, or which has a reduced affinity to an Fc receptor.

A “native-sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature.Native-sequence human Fc regions include, for example, a native-sequencehuman IgG1 Fc region (non-A and A allotypes); native-sequence human IgG2Fc region; native-sequence human IgG3 Fc region; and native-sequencehuman IgG4 Fc region, as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence that differs fromthat of a native-sequence Fc region by virtue of at least one amino acidmodification, for example, one or more (e.g., two or more, three ormore, four or more) amino acid substitution(s). Illustratively, in someembodiments, the variant Fc region has at least one amino acidsubstitution compared to a native-sequence Fc region or to the Fc regionof a parent polypeptide, e.g., from about one to about ten amino acidsubstitutions, e.g., from about one to about five amino acidsubstitutions in a native-sequence Fc region or in the Fc region of theparent polypeptide. In some embodiments, the variant Fc region hereinwill possess at least about 80% homology with a native-sequence Fcregion and/or with an Fc region of a parent polypeptide, e.g., at leastabout 85% homology therewith, e.g., at least about 90% homologytherewith, e.g., at least about 95% homology therewith, e.g., at leastabout 99% homology therewith.

As used herein, “heterodimerizing alterations” refer to alterations inthe A and B chains of an Fc region (i.e., the two chains comprising theFc region, wherein one chain is referred to herein as the “A” chain andthe other is referred to herein as the “B” chain) that facilitate theformation of heterodimeric Fc regions, that is, Fc regions in which theA chain and the B chain of the Fc region do not have identical aminoacid sequences. In some embodiments, heterodimerizing alterations can beasymmetric, that is, an A chain having a certain alteration can pairwith a B chain having a different alteration. These alterationsfacilitate heterodimerization and disfavor homodimerization. Whetherhetero- or homo-dimers have formed can be assessed, for example, by sizedifferences as determined by polyacrylamide gel electrophoresis insituations where one polypeptide chain is a dummy Fc and the other is anscFv-Fc. One non-limiting example of such paired heterodimerizingalterations are the so-called “knobs and holes” substitutions. See,e.g., U.S. Pat. No. 7,695,936 and U.S. Patent Application PublicationNo. 2003/0078385. As used herein, an Fc region that comprises one pairof knobs and holes substitutions, comprises one substitution in the Achain and another in the B chain. For example, the following knobs andholes substitutions in the A and B chains of an IgG1 Fc region have beenfound to increase heterodimer formation as compared with that found withunmodified A and B chains and may be employed in non-limitingembodiments of this disclosure: 1) Y407T in one chain and T366Y in theother; 2) Y407A in one chain and T366W in the other; 3) F405A in onechain and T394W in the other; 4) F405W in one chain and T394S in theother; 5) Y407T in one chain and T366Y in the other; 6) T366Y and F405Ain one chain and T394W and Y407T in the other; 7) T366W and F405W in onechain and T394S and Y407A in the other; 8) F405W and Y407A in one chainand T366W and T394S in the other; and 9) T366W in one polypeptide of theFe and T366S, L368A, and Y407V in the other. Alternatively, or inaddition to such alterations, substitutions creating new disulfidebridges can facilitate heterodimer formation. See, e.g., U.S. PatentApplication Publication No. 2003/0078385. Such alterations in an IgG1 Fcregion include, but are not limited to, the following substitutions:Y349C in one Fc polypeptide chain and S354C in the other; Y349C in oneFc polypeptide chain and E356C in the other; Y349C in one Fc polypeptidechain and E357C in the other; L351C in one Fc polypeptide chain andS354C in the other; T394C in one Fc polypeptide chain and E397C in theother; or D399C in one Fc polypeptide chain and K392C in the other.Additionally or alternatively, substitutions changing the charge of aone or more residue(s), for example, in the CH3-CH3 interface, canenhance heterodimer formation, as described, for example, in WO2009/089004, which is incorporated by reference herein. Suchsubstitutions are referred to herein as “charge pair substitutions,” andan Fc region comprising one pair of charge pair substitutions comprisesone substitution in the A chain and a different substitution in the Bchain. Non-limiting examples of charge pair substitutions include thefollowing: 1) K409D or K409E in one chain plus D399K or D399R in theother; 2) K392D or K392E in one chain plus D399K or D399R in the other;3) K439D or K439E in one chain plus E356K or E356R in the other; and 4)K370D or K370E in one chain plus E357K or E357R in the other. Inaddition, the substitutions R355D, R355E, K360D, or K360R in both chainscan stabilize heterodimers when used with other heterodimerizingalterations. Specific charge pair substitutions can be used either aloneor with other charge pair substitutions. Specific examples of singlepairs of charge pair substitutions and combinations thereof include thefollowing: 1) K409E in one chain plus D399K in the other; 2) K409E inone chain plus D399R in the other; 3) K409D in one chain plus D399K inthe other; 4) K409D in one chain plus D399R in the other; 5) K392E inone chain plus D399R in the other; 6) K392E in one chain plus D399K inthe other; 7) K392D in one chain plus D399R in the other; 8) K392D inone chain plus D399K in the other; 9) K409D and K360D in one chain plusD399K and E356K in the other; 10) K409D and K370D in one chain plusD399K and E357K in the other; 11) K409D and K392D in one chain plusD399K, E356K, and E357K in the other; 12) K409D and K392D on one chainand D399K on the other; 13) K409D and K392D on one chain plus D399K andE356K on the other; 14) K409D and K392D on one chain plus D399K andD357K on the other; 15) K409D and K370D on one chain plus D399K andD357K on the other; 16) D399K on one chain plus K409D and K360D on theother; and 17) K409D and K439D on one chain plus D399K and E356K on theother. Any of these heterodimerizing alterations can be used inpolypeptides comprising variant Fc regions as described herein.

In some non-limiting embodiments, variant Fc sequences may include threeamino acid substitutions in the CH2 region to reduce FcγRI binding at EUindex positions 234, 235, and 237 (see Duncan et al., (1988) Nature332:563). Two amino acid substitutions in the complement C1q bindingsite at EU index positions 330 and 331 reduce complement fixation (seeTao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J.Exp. Med. 173:1483 (1991)). Substitution into human IgG1 or IgG2residues at positions 233-236 and IgG4 residues at positions 327, 330and 331 greatly reduces ADCC and CDC (see, for example, Armour K L. etal., 1999 Eur J Immunol. 29(8):2613-24; and Shields R. L. et al., 2001.J Biol Chem. 276(9):6591-604). The human IgG4 Fc amino acid sequence(UniProtKB No. P01861) is provided herein as SEQ ID NO: 76. SilencedIgG1 is described, for example, in Boesch, A. W., et al., “Highlyparallel characterization of IgG Fc binding interactions.” MAbs, 2014.6(4): p. 915-27, the disclosure of which is incorporated herein byreference in its entirety.

Other Fc variants are possible, including, without limitation, one inwhich a region capable of forming a disulfide bond is deleted, or inwhich certain amino acid residues are eliminated at the N-terminal endof a native Fc, or a methionine residue is added thereto. Thus, in someembodiments, one or more Fc portions of an antibody can comprise one ormore mutations in the hinge region to eliminate disulfide bonding. Inyet another embodiment, the hinge region of an Fc can be removedentirely. In still another embodiment, an antibody can comprise an Fcvariant.

Further, an Fc variant can be constructed to remove or substantiallyreduce effector functions by substituting (mutating), deleting, oradding amino acid residues to effect complement binding or Fc receptorbinding. For example, and not by way of limitation, a deletion may occurin a complement-binding site, such as a C1q-binding site. Techniques forpreparing such sequence derivatives of the immunoglobulin Fc fragmentare disclosed in International Patent Publication Nos. WO 97/34631 andWO 96/32478. In addition, the Fc domain may be modified byphosphorylation, sulfation, acylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like.

Antibodies with reduced effector function include, but are not limitedto, those with substitution of one or more of Fc region residues 238,265, 269, 270, 297, 327 and 329 according to EU numbering (see, e.g.,U.S. Pat. No. 6,737,056). In some embodiments, variant Fc regions withreduced effector function comprise substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327 according to EU numbering,including the so-called “DANA” Fc mutant with substitution of residues265 and 297 to alanine according to EU numbering (i.e., D265A and N297Aaccording to EU numbering) (see, e.g., U.S. Pat. No. 7,332,581). In someembodiments, the variant Fc region with reduced effector functioncomprises the following two amino acid substitutions: D265A and N297A.

In some embodiments, effector function is reduced through a mutation ina constant region that eliminates glycosylation, e.g., an “effector-lessmutation.” In some embodiments, the effector-less mutation is an N297Aor a DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J.Biol. Chem. 276 (9): 6591-6604 (2001). In some embodiments, theeffector-less mutation is an N297G or a DANG mutation (D265A+N297G) inthe CH2 region. In some embodiments, the variant Fc region lacksglycosylation at N297, e.g., the variant Fc region is a variant Fcregion lacking glycosylation at N297 as described in InternationalPatent Publication No. WO 2014/153063, which is incorporated byreference herein. Alternatively, additional mutations resulting inreduced or eliminated effector function include: K322A and L234A/L235A(LALA). Alternatively, effector function can be reduced or eliminatedthrough production techniques, such as expression in host cells that donot glycosylate (e.g., E. coli) or in host cells which result in analtered glycolsylation pattern that is ineffective or less effective atpromoting effector function (e.g., Shinkawa et al., J. Biol. Chem.278(5): 3466-3473 (2003)).

In some embodiments, the proline at position 329 (EU numbering) (P329)of a wild-type human Fc region is substituted with glycine or arginineor an amino acid residue large enough to destroy the proline sandwichwithin the Fc/Fcγ receptor interface, that is formed between the P329 ofthe Fc and tryptophan residues W87 and W110 of FcgRIII (Sondermann etal., Nature 406, 267-273 (20 Jul. 2000)). In some further embodiments,at least one further amino acid substitution in the Fc variant region isS228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In someembodiments, the at least one further amino acid substitution is L234Aand L235A of the human IgG1 Fc region or S228P and L235E of the humanIgG4 Fc region, all according to EU numbering (see, e.g., U.S. Pat. No.8,969,526, which is incorporated by reference in its entirety).

In some embodiments, the variant Fc region has P329 of the human IgG Fcregion substituted with glycine, wherein the variant Fc region comprisesat least two further amino acid substitutions at L234A and L235A of thehuman IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, andwherein the residues are numbered according to the EU numbering (see,e.g., U.S. Pat. No. 8,969,526). In some embodiments, the variant Fcregion comprising the P329G, L234A and L235A (EU numbering)substitutions exhibits a reduced affinity to the human FcγRIIIA andFcγRIIA.

In some embodiments, the variant Fc region comprises a triple mutation:an amino acid substitution at position P329, a L234A, and a L235Amutation according to EU numbering (P329/LALA) (see, e.g., U.S. Pat. No.8,969,526). In some embodiments, the variant Fc region comprises thefollowing amino acid substitutions: P329G, L234A, and L235A according toEU numbering.

In some embodiments, an antibody comprises a variant human IgG4 CH3domain sequence comprising a T366W mutation, which can optionally bereferred to herein as an IgG4 CH3 knob sequence. In some embodiments, anantibody comprises a variant human IgG4 CH3 domain sequence comprising aT366S mutation, an L368A mutation, and a Y407V mutation, which canoptionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4CH3 mutations described herein can be utilized in any suitable manner soas to place a “knob” on a first heavy chain constant region of a firstmonomer in an antibody dimer, and a “hole” on a second heavy chainconstant region of a second monomer in an antibody dimer, therebyfacilitating proper pairing (heterodimerization) of the desired pair ofheavy chain polypeptide subunits in the antibody.

In some embodiments, an antibody comprises a heavy chain polypeptidesubunit comprising a variant human IgG4 Fc region comprising an S228Pmutation, an F234A mutation, an L235A mutation, and a T366W mutation(knob). In some embodiments, an antibody comprises a heavy chainpolypeptide subunit comprising a variant human IgG4 Fc region comprisingan S228P mutation, an F234A mutation, an L235A mutation, a T366Smutation, an L368A mutation, and a Y407V mutation (hole).

The term “Fc-region-comprising antibody” refers to an antibody thatcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, an antibody having anFc region according to this disclosure can comprise an antibody with orwithout K447.

Aspects of the disclosure include antibodies comprising a heavychain-only variable region in a monovalent or bivalent configuration. Asused herein, the term “monovalent configuration”, as used in referenceto a heavy chain-only variable region domain, means that only one heavychain-only variable region domain is present, having a single bindingsite. In contrast, the term “bivalent configuration” as used inreference to a heavy chain-only variable region domain means that twoheavy chain-only variable region domains are present (each having asingle binding site), and are connected by a linker sequence.Non-limiting examples of linker sequences are discussed further herein,and include, without limitation, GS linker sequences of various lengths.When a heavy chain-only variable region is in a bivalent configuration,each of the two heavy chain-only variable region domains can bind to thesame antigen, or to different antigens (e.g., to different epitopes onthe same protein; to two different proteins, etc.). However, unlessspecifically noted otherwise, a heavy chain-only variable region denotedas being in a “bivalent configuration” is understood to contain twoidentical heavy chain-only variable region domains, connected by alinker sequence, wherein each of the two identical heavy chain-onlyvariable region domains binds to the same target antigen.

Aspects of the disclosure include antibodies having multi-specificconfigurations, which include, without limitation, bispecific,trispecific, etc. configurations. A large variety of methods and proteinconfigurations are known and used in bispecific monoclonal antibodies(BsMAB), tri-specific antibodies, etc.

Various methods for the production of multivalent artificial antibodieshave been developed by recombinantly fusing variable domains of two ormore antibodies. In some embodiments, a first and a secondantigen-binding domain on a polypeptide are connected by a polypeptidelinker. One non-limiting example of such a polypeptide linker is a GSlinker, having an amino acid sequence of four glycine residues, followedby one serine residue, and wherein the sequence is repeated n times,where n is an integer ranging from 1 to about 10 (SEQ ID NO: 68), suchas 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkersinclude (SEQ ID NO: 49), (n=1) and GGGGSGGGGS (SEQ ID NO: 50) (n=2).Other suitable linkers can also be used, and are described, for example,in Chen et al., Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-69, thedisclosure of which is incorporated herein by reference in its entirety.

The term “three-chain antibody-like molecule” or “TCA” is used herein torefer to antibody-like molecules comprising, consisting essentially of,or consisting of three polypeptide subunits, two of which comprise,consist essentially of, or consist of one heavy and one light chain of amonoclonal antibody, or functional antigen-binding fragments of suchantibody chains, comprising an antigen-binding region and at least oneCH domain. This heavy chain/light chain pair has binding specificity fora first antigen. The third polypeptide subunit comprises, consistsessentially of, or consists of a heavy-chain only antibody comprising anFc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absenceof a CH1 domain, and one or more antigen binding domains (e.g., twoantigen binding domains) that binds an epitope of a second antigen or adifferent epitope of the first antigen, where such binding domain isderived from or has sequence identity with the variable region of anantibody heavy or light chain. Parts of such variable region may beencoded by V_(H) and/or V_(L) gene segments, D and J_(H) gene segments,or J_(L) gene segments. The variable region may be encoded by rearrangedV_(H)DJ_(H), V_(L)DJ_(H), V_(H)J_(L), or V_(L)J_(L) gene segments.

A TCA binding compound makes use of a “heavy chain only antibody” or“heavy chain antibody” or “heavy chain polypeptide” which, as usedherein, mean a single chain antibody comprising heavy chain constantregions CH2 and/or CH3 and/or CH4 but no CH1 domain. In one embodiment,the heavy chain antibody is composed of an antigen-binding domain, atleast part of a hinge region and CH2 and CH3 domains. In anotherembodiment, the heavy chain antibody is composed of an antigen-bindingdomain, at least part of a hinge region and a CH2 domain. In a furtherembodiment, the heavy chain antibody is composed of an antigen-bindingdomain, at least part of a hinge region and a CH3 domain. Heavy chainantibodies in which the CH2 and/or CH3 domain is truncated are alsoincluded herein. In a further embodiment, the heavy chain is composed ofan antigen binding domain, and at least one CH (CH1, CH2, CH3, or CH4)domain but no hinge region. The heavy chain only antibody can be in theform of a dimer, in which two heavy chains are disulfide bonded orotherwise covalently or non-covalently attached to each other, and canoptionally include an asymmetric interface (e.g., a knobs-in-holes (KiH)interface) between one or more of the CH domains to facilitate properpairing between polypeptide chains. The heavy-chain antibody may belongto the IgG subclass, but antibodies belonging to other subclasses, suchas IgM, IgA, IgD and IgE subclass, are also included herein. In aparticular embodiment, the heavy chain antibody is of the IgG1, IgG2,IgG3, or IgG4 subtype, in particular the IgG1 subtype or the IgG4subtype. Non-limiting examples of a TCA binding compound are describedin, for example, WO2017/223111 and WO2018/052503, the disclosures ofwhich are incorporated herein by reference in their entirety.

Aspects of the disclosure include antibodies that comprise a heavychain-only variable region that is paired with a light chain variableregion (VL). In some embodiments, a light chain variable region thatpairs with a heavy chain-only variable region is referred to as a “fixedlight chain” variable region. In certain embodiments, an antibodycomprises two heavy chain-only variable regions, each of which is pairedwith a fixed light chain variable region. In some embodiments, a fixedlight chain variable region sequence is connected to a light chainconstant region sequence to form a full-length antibody light chainpolypeptide. In some embodiments, an antibody comprises two full lengthheavy chain polypeptides and two full length light chain polypeptides.In certain embodiments, the full-length heavy chain polypeptidescomprise different sequences, whereas the full length light chainpolypeptides comprise the same sequence (e.g., the two full-length lightchain polypeptides are identical).

Heavy-chain antibodies constitute about one fourth of the IgG antibodiesproduced by the camelids, e.g., camels and llamas (Hamers-Casterman C.,et al. Nature. 363, 446-448 (1993)). These antibodies are formed by twoheavy chains but are devoid of light chains. As a consequence, thevariable antigen binding part is referred to as the VHH domain, and itrepresents the smallest naturally occurring, intact, antigen-bindingsite, being only around 120 amino acids in length (Desmyter, A., et al.J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with ahigh specificity and affinity can be generated against a variety ofantigens through immunization (van der Linden, R. H., et al. Biochim.Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be readilycloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol.78, 11-21 (2000)). Their levels of expression, solubility and stabilityare significantly higher than those of classical F(ab) or Fv fragments(Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks havealso been shown to have a single VH-like domain in their antibodies,termed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003);Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley etal., Molecular Immunology 40, 25-33 (2003).)

The terms “IL2” and “IL-2” as used interchangeably herein refer tointerleukin-2, which is a 15.5 to 16 kDa cytokine signaling proteinmolecule that regulates the activity of certain immune cells by bindingto IL2 receptor complexes expressed by lymphocytes. The term “IL2”includes an IL2 protein of any human and non-human animal species, andspecifically includes human IL2 as well as IL2 of non-human mammals. Thehuman IL-2 sequence (UniProtKB No. P60568) is provided herein as SEQ IDNO: 41. The term “human IL2” as used herein includes any variants,isoforms, and species homologs of human IL2, regardless of its source ormode of preparation. Thus, “human IL2” includes human IL2 naturallyexpressed by cells and IL2 expressed on cells transfected with the humanIL2 gene.

The terms “IL2R”, “IL-2R”, “IL2 receptor”, and “IL-2 receptor”, as usedinterchangeably herein refer generally to the IL2 receptor complex,which is composed of three polypeptide subunits, or chains, referred toas the alpha, A, or α chain, the beta, B, or β chain, and the gamma, G,or γ chain. The term “IL2R” includes any IL2R protein or any subunit ofthe IL2 receptor complex, of any human and non-human animal species, andspecifically includes human IL2R as well as IL2R of non-human mammals.The term “human IL2R” as used herein includes any variants, isoforms,and species homologs of human IL2R, regardless of its source or mode ofpreparation. Thus, “human IL2R” includes human IL2R naturally expressedby cells and IL2R expressed on cells transfected with the human IL2Rgene.

The term “IL2RA” is also referred to as CD25, and the human IL2RAsequence (UniProtKB No. P01589) is provided herein as SEQ ID NO: 38.

The term “IL2RB” IL-2RB is also referred to as CD122, and the humanIL2RB sequence (UniProtKB No. P14784) is provided herein as SEQ ID NO:39.

The term “IL2RG” IL-2RG is also referred to as CD132, and the humanIL2RG sequence (UniProtKB No. P31785) is provided herein as SEQ ID NO:40.

The terms “anti-IL2R heavy chain-only antibody,” “IL2R heavy chain-onlyantibody,” “anti-IL2R heavy chain antibody,” and “IL2R heavy chainantibody” are used herein interchangeably to refer to a heavy chain-onlyantibody as hereinabove defined, immunospecifically binding to IL2R,including human IL2R, as hereinabove defined. The definition includes,without limitation, human heavy chain antibodies produced by transgenicanimals, such as transgenic rats or transgenic mice expressing humanimmunoglobulin, including UniRats™ producing human anti-IL2R UniAb™antibodies, as hereinabove defined.

The term “agonist” as used herein refers to a molecule that causes anincrease in a function or activity as compared to the same function oractivity in the absence of the molecule. An “agonist” of a signalingpathway is therefore a molecule whose presence causes an increase in afunction or activity of the signaling pathway. The term “agonize” asused herein refers to causing an increase in a function or activity. Insome embodiments, the agonist function of an antibody may be determinedusing an assay described herein.

The term “antagonist” as used herein refers to a molecule that causes adecrease in a function or activity as compared to the same function oractivity in the absence of the molecule. An “antagonist” of a signalingpathway is therefore a molecule whose presence causes a decrease in afunction or activity of the signaling pathway. The term “antagonize” asused herein refers to causing a decrease in a function or activity.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, e.g., more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or nonreducing conditions usingCoomassie blue or, e.g., silver stain. Isolated antibody includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least one purificationstep.

Antibodies of the disclosure include multi-specific antibodies.Multi-specific antibodies have more than one binding specificity. Theterm “multi-specific” specifically includes “bispecific” and“trispecific,” as well as higher-order independent specific bindingaffinities, such as higher-order polyepitopic specificity, as well astetravalent antibodies and antibody fragments. The terms “multi-specificantibody,” “multi-specific heavy chain-only antibody,” “multi-specificheavy chain antibody,” and “multi-specific UniAb™” are used herein inthe broadest sense and cover all antibodies with more than one bindingspecificity. The multi-specific heavy chain anti-IL2R antibodies of thepresent disclosure specifically include antibodies immunospecificallybinding to two or more non-overlapping epitopes on an IL2R protein, suchas a human IL2RA, IL2RB and/or IL2RG protein. The multi-specific heavychain anti-IL2R antibodies of the present disclosure also specificallyinclude antibodies immunospecifically binding to an epitope on an IL2Rprotein, such as human IL2RB, and to an epitope on a different protein,such as, for example, an IL2RG protein, such as human IL2RG.

Antibodies of the disclosure include monospecific antibodies, having onebinding specificity. Monospecific antibodies specifically includeantibodies comprising a single binding specificity, as well asantibodies comprising more than one binding unit having the same bindingspecificity. The terms “monospecific antibody,” “monospecific heavychain-only antibody,” “monospecific heavy chain antibody,” and“monospecific UniAb™” are used herein in the broadest sense and coverall antibodies with one binding specificity. The monospecific heavychain anti-IL2R antibodies of the present disclosure specificallyinclude antibodies immunospecifically binding to one epitope on an IL2Rprotein, such as a human IL2R protein, or subunit thereof (e.g., a humanIL2RA, IL2RB, or IL2RG protein). The monospecific heavy chain anti-IL2Rantibodies of the present disclosure also specifically includeantibodies having more than one binding unit (e.g., multivalentantibodies) immunospecifically binding to an epitope on an IL2R protein,such as human IL2R. For example, a monospecific antibody in accordancewith embodiments of the disclosure can include a heavy chain variableregion comprising two antigen-binding domains, wherein eachantigen-binding domain binds to the same epitope on an IL2R protein(i.e., an IL2RA, IL2RB, or IL2RG protein).

An “epitope” is the site on the surface of an antigen molecule to whicha single antibody molecule binds. Generally, an antigen has several ormany different epitopes and reacts with many different antibodies. Theterm specifically includes linear epitopes and conformational epitopes.

“Epitope mapping” is the process of identifying the binding sites, orepitopes, of antibodies on their target antigens. Antibody epitopes maybe linear epitopes or conformational epitopes. Linear epitopes areformed by a continuous sequence of amino acids in a protein.Conformational epitopes are formed of amino acids that are discontinuousin the protein sequence, but which are brought together upon folding ofthe protein into its three-dimensional structure.

“Polyepitopic specificity” refers to the ability to specifically bind totwo or more different epitopes on the same or different target(s). Asnoted above, the present disclosure specifically includes anti-IL2Rheavy chain antibodies with polyepitopic specificities, i.e., anti-IL2Rheavy chain antibodies binding to one or more non-overlapping epitopeson a first IL2R protein, such as a human IL2RB; and anti-IL2R heavychain antibodies binding to one or more epitopes on a first IL2R protein(e.g., an IL2RB protein) and to an epitope on a different IL2R protein,such as, for example, an IL2RG protein. The term “non-overlappingepitope(s)” or “non-competitive epitope(s)” of an antigen is definedherein to mean epitope(s) that are recognized by one member of a pair ofantigen-specific antibodies but not the other member. Pairs ofantibodies, or antigen-binding regions targeting the same antigen on amulti-specific antibody, recognizing non-overlapping epitopes, do notcompete for binding to that antigen and are able to bind that antigensimultaneously.

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels.

The term “valent” as used herein refers to a specified number of bindingsites in an antibody molecule.

A “monovalent” antibody has one binding site. Thus, a monovalentantibody is also monospecific.

A “multi-valent” antibody has two or more binding sites. Thus, the terms“bivalent,” “trivalent,” and “tetravalent” refer to the presence of twobinding sites, three binding sites, and four binding sites,respectively. Thus, a bispecific antibody according to the disclosure isat least bivalent and may be trivalent, tetravalent, or otherwisemulti-valent. A bivalent antibody in accordance with embodiments of thedisclosure may have two binding sites to the same epitope (i.e.,bivalent, monoparatopic), or to two different epitopes (i.e., bivalent,biparatopic).

A large variety of methods and protein configurations are known and usedfor the preparation of bispecific monoclonal antibodies (BsMAB),tri-specific antibodies, and the like.

The term “human antibody” is used herein to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies herein may include amino acid residuesnot encoded by human germline immunoglobulin sequences, e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo. The term “human antibody” specifically includes heavychain-only antibodies having human heavy chain variable regionsequences, produced by transgenic animals, such as transgenic rats ormice, in particular UniAbs™ produced by UniRats™, as defined above.

By a “chimeric antibody” or a “chimeric immunoglobulin” is meant animmunoglobulin molecule comprising amino acid sequences from at leasttwo different Ig loci, e.g., a transgenic antibody comprising a portionencoded by a human Ig locus and a portion encoded by a rat Ig locus.Chimeric antibodies include transgenic antibodies with non-humanFc-regions or artificial Fc-regions, and human idiotypes. Suchimmunoglobulins can be isolated from animals of the disclosure that havebeen engineered to produce such chimeric antibodies.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Some effectorcells express specific Fc receptors and carry out specific immunefunctions. In some embodiments, an effector cell such as a naturalkiller cell is capable of inducing antibody-dependent cellularcytotoxicity (ADCC). For example, monocytes and macrophages, whichexpress FcR, are involved in specific killing of target cells andpresenting antigens to other components of the immune system, or bindingto cells that present antigens. In some embodiments, an effector cellmay phagocytose a target antigen or target cell.

“Human effector cells” are leukocytes which express receptors such asT-cell receptors or FcRs and perform effector functions. For example, insome embodiments, the cells express at least FcγRIII and perform ADCCeffector function. Examples of human leukocytes which mediate ADCCinclude natural killer (NK) cells, monocytes, cytotoxic T-cells, andneutrophils. The effector cells may be isolated from a native sourcethereof, e.g., from blood or PBMCs as described herein.

The term “immune cell” is used herein in the broadest sense, including,without limitation, cells of myeloid or lymphoid origin, for instancelymphocytes (such as B-cells and T-cells including cytolytic T-cells(CTLs)), killer cells, natural killer (NK) cells, macrophages,monocytes, eosinophils, polymorphonuclear cells, such as neutrophils,granulocytes, mast cells, and basophils.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g., B-cell receptor; BCR), etc.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g., an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods202:163 (1996), may be performed.

“Binding affinity” refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art. Low-affinity antibodies generally bind antigen slowlyand tend to dissociate readily, whereas high-affinity antibodiesgenerally bind antigen faster and tend to remain bound.

As used herein, the “Kd” or “Kd value” refers to a dissociation constantdetermined by BioLayer Interferometry, using an Octet QK384 instrument(Fortebio Inc., Menlo Park, CA) in kinetics mode. For example,anti-mouse Fc sensors are loaded with mouse-Fc fused antigen and thendipped into antibody-containing wells to measure concentration dependentassociation rates (kon). Antibody dissociation rates (koff) are measuredin the final step, where the sensors are dipped into wells containingbuffer only. The Kd is the ratio of koff/kon. (For further details see,Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800,2009).

The terms “treatment,” “treating,” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease. Thetherapeutic agent may be administered before, during, or after the onsetof disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy may be administered during the symptomatic stage ofthe disease, and in some cases after the symptomatic stage of thedisease.

A “therapeutically effective amount” is intended for an amount of activeagent which is necessary to impart therapeutic benefit to a subject. Forexample, a “therapeutically effective amount” is an amount whichinduces, ameliorates, or otherwise causes an improvement in thepathological symptoms, disease progression, or physiological conditionsassociated with a disease or which improves resistance to a disorder.

The term “mediated by activation of IL2R signaling in immune cells”broadly refers to any disease or disorder in which the IL2/IL2Rsignaling pathway is associated with or involved with one or morepathological processes that are characteristic of the disease ordisorder. Such disorders include, but are not limited to, infectiousdiseases, autoimmune disorders (e.g., Crohn's disease, multiplesclerosis), cancer, inflammatory diseases (e.g., arthritis), or diseasesor disorders associated with deficient IL-2-mediated signaling,deficient T cell proliferation, or T cell dysfunction.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having cancer, individuals with autoimmune diseases, withpathogen infections, and the like. Subjects may be human, but alsoinclude other mammals, particularly those mammals useful as laboratorymodels for human disease, e.g., mouse, rat, etc.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile. “Pharmaceuticallyacceptable” excipients (e.g., vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

A “sterile” formulation is aseptic or free or essentially free from allliving microorganisms and their spores. A “frozen” formulation is one ata temperature below 0° C.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. In some embodiments, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage. The storage period is generallyselected based on the intended shelf-life of the formulation. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301.Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. Stability can be evaluated qualitatively and/or quantitativelyin a variety of different ways, including evaluation of aggregateformation (for example, using size exclusion chromatography, bymeasuring turbidity, and/or by visual inspection); by assessing chargeheterogeneity using cation exchange chromatography, image capillaryisoelectric focusing (icIEF) or capillary zone electrophoresis;amino-terminal or carboxy-terminal sequence analysis; mass spectrometricanalysis; SDS-PAGE analysis to compare reduced and intact antibody;peptide map (for example tryptic or LYS-C) analysis; evaluatingbiological activity or antigen binding function of the antibody; etc.Instability may involve any one or more of: aggregation, deamidation(e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization(e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g.,hinge region fragmentation), succinimide formation, unpairedcysteine(s), N-terminal extension, C-terminal processing, glycosylationdifferences, etc.

Anti-IL2R Antibodies

The present disclosure provides various families of antibodies that bindto human IL2R. Aspects of the disclosure include closely relatedantibody families whose members bind to a particular IL2R subunit, orchain, e.g., that bind to IL2RB or IL2RG, or a combination thereof.

In some embodiments, an anti-IL2RB antibody comprises CDR sequenceshaving the following sequence formulae. An X indicates a variable aminoacid, which may, in some embodiments, be the specific amino acid listedbelow:

-   -   CDR1 (IL2RB_F09)        -   G G S I S S S X1 W (SEQ ID NO:26)            where X1 is D or N;    -   CDR2 (IL2RB_F09)        -   I X2 H S G S T (SEQ ID NO: 27)            where X2 is D or S; and    -   CDR3 (IL2RB_F09)        -   X3 R G X4 W E L X5 D A F D I (SEQ ID NO: 28)            where:    -   X3 is G or A;    -   X4 is S or Q; and    -   X5 is S or T.

In some embodiments, an anti-IL2RB antibody comprises any combination ofCDR1, and CDR2 and CDR3 sequences comprising the sequence formulae ofSEQ ID NOs: 26, 27, and 28, respectively. Antibodies of this family canbe referred to herein as IL2RB_F09 antibodies.

In some embodiments, an anti-IL2RB antibody comprises CDR sequenceshaving the following sequence formulae. An X indicates a variable aminoacid, which may, in some embodiments, be the specific amino acid listedbelow:

-   -   CDR1 (IL2RB_F18)

G F T F S X1 Y G (SEQ ID NO: 29)

where X1 is S or T;

-   -   CDR2 (IL2RB_F18)        -   I S Y D G S N X2 (SEQ ID NO: 30)            where X2 is K or R; and    -   CDR3 (IL2RB_F18)        -   A R D L D Y D X3 L T G D P V G G F D I (SEQ ID NO: 31)            where X3 is V or I.

In some embodiments, an anti-IL2RB antibody comprises any combination ofCDR1, CDR2, and CDR3 sequences comprising the sequence formulae of SEQID NOs: 29, 30, and 31, respectively. Antibodies of this family can bereferred to herein as IL2RB_F18 antibodies.

Antibodies in accordance with embodiments of the disclosure that bind toIL2RB can comprise a set of CDR sequences as defined herein andexemplified by the provided heavy chain CDR1, CDR2 and CDR3 sequencesset forth in Table 1, and the heavy chain variable region (VH) sequencesset forth in Table 2. These antibodies provide a number of benefits thatcontribute to utility as clinically therapeutic agent(s). The antibodiesinclude members with a range of binding affinities, allowing theselection of a specific sequence with a desired binding affinity.

TABLE 1 Anti-IL2RB Heavy Chain Antibody Unique CDR Amino Acid SequencesClone ID No. Family ID No. CDR1 CDR2 CDR3 387205 IL2RB_F09C GGSISSSDWIDHSGST GRGSWELSDAFDI (SEQ ID NO: 1) (SEQ ID NO: 4) (SEQ ID NO: 7)387172 IL2RB_F09G GGSISSSDW IDHSGST ARGSWELTDAFDI (SEQ ID NO: 1) (SEQ IDNO: 4) (SEQ ID NO: 8) 387111 IL2RB_F09K GGSISSSNW ISHSGST GRGSWELTDAFDI(SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 9) 388252 IL2RB_F18E GFTFSSYGISYDGSNK ARDLDYDVLTGDPVGGFDI (SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO:10)

TABLE 2 Anti-IL2RB Heavy Chain Antibody Variable Region Amino AcidSequences Clone ID No. Family ID No. VH sequence 387205 IL2RB_F09CQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWIGEIDHSGSTNYNPSLMSRVTISVDKSKNQFSLKLSSVTAADTAVYFCGRGSWELSDAFDIRGQGTLVTVSS (SEQ ID NO: 11) 387172 IL2RB_F09GQVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVRQPPGKGLEWIGEIDHSGSTNYNPSLMSRVTISVDKSKNQFSLKLSSVTAADTAVYFCARGSWELTDAFDIRGQGTLVTVSS (SEQ ID NO: 12) 387111 IL2RB_F09KQVQLQESSPGLVKPSETLSLTCTVSGGSISSSNWWSWVRQPPGKGLEWIGEISHSGSTNYNPSLKSRVTISVDKSKNQFSLRLSSVTAADTAVYFCGRGSWELTDAFDIRGQGTLVTVSS (SEQ ID NO: 13) 388252 IL2RB_F18EQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREWVAVISYDGSNKYYTDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYCARDLDYDVLTGDPVGGFDIWGQGTLVTV SS (SEQ ID NO: 14)

In some embodiments, an anti-IL2RG antibody comprises CDR sequenceshaving the following sequence formulae. An X indicates a variable aminoacid, which may, in some embodiments, be the specific amino acid listedbelow:

-   -   CDR1 (IL2RG_F16)        -   G F X1 X2 X3 X4 Y Y (SEQ ID NO: 32)            where:    -   X1 is T or I;    -   X2 is F or V;    -   X3 is S, N, or G; and    -   X4 is D or N;    -   CDR2 (IL2RG_F16)        -   I S X5 S G X6 X7 I (SEQ ID NO: 33)            where:    -   X5 is S or N;    -   X6 is D, S, G, or N; and    -   X7 is T or I; and    -   CDR3 (IL2RG_F16)        -   ARGDAVSITGDY (SEQ ID NO: 20).

In some embodiments, an anti-IL2RG antibody comprises any combination ofCDR1, and CDR2 and CDR3 sequences comprising the sequence formulae ofSEQ ID NOs: 32, 33, and 34, respectively. Antibodies of this family canbe referred to herein as IL2RG_F16 antibodies.

In some embodiments, an anti-IL2RG antibody comprises a CDR1 sequencecomprising GFTFSDYY (SEQ ID NO: 15), a CDR2 (IL2RG_F18) sequencecomprising ISSSGTTT (SEQ ID NO: 19), and a CDR3 (IL2RG_F18) sequencecomprising ARGAAVAPGFDS (SEQ ID NO: 21). Antibodies of this family canbe referred to herein as IL2RG_F18 antibodies.

Antibodies in accordance with embodiments of the disclosure that bind toIL2RG comprise a set of CDR sequences as defined herein and exemplifiedby the provided heavy chain CDR1, CDR2, and CDR3 sequences set forth inTable 3, and the heavy chain variable region (VH) sequences set forth inTable 4. This family of antibodies provides a number of benefits thatcontribute to utility as clinically therapeutic agent(s). The antibodiesinclude members with a range of binding affinities, allowing theselection of a specific sequence with a desired binding affinity.

TABLE 3 Anti-IL2RG Heavy Chain Antibody CDR1, CDR2 and CDR3 Amino AcidSequences Clone ID No. Family ID No. CDR1 CDR2 CDR3 363256 IL2RG_F16AGFTFSDYY ISSSGDTI ARGDAVSITGDY (SEQ ID NO: 15) (SEQ ID NO: 17) (SEQ IDNO: 20) 363544 IL2RG_F16B GFTFSDYY ISSSGSTI ARGDAVSITGDY (SEQ ID NO: 15)(SEQ ID NO: 18) (SEQ ID NO: 20) 388582 IL2RG_F16C GFTFNDYY ISSSGSTIARGDAVSITGDY (SEQ ID NO: 16) (SEQ ID NO: 18) (SEQ ID NO: 20) 363435IL2RG_F18A GFTFSDYY ISSSGTTT ARGAAVAPGFDS (SEQ ID NO: 15) (SEQ ID NO:19) (SEQ ID NO: 21)

TABLE 4 Anti-IL2RG Heavy Chain Antibody Variable Region Amino AcidSequences Clone ID No. Family ID No. VH Sequence 363256 IL2RG_F16AQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSSISSSGDTIYYADSVQGRFTLSRDNAENSLFLQMNSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSS (SEQ ID NO: 22) 363544 IL2RG_F16BQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSS (SEQ ID NO: 23) 388582 IL2RG_F16CQVQLVESGGGLVKPGGSLRLSCAASGFTFNDYYMSWIRQAPGKGLEWVSHISSSGSTIYYADSVKGRFTVSRDNANNSLYLQMHSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSS (SEQ ID NO: 24) 363435 IL2RG_F18AQVQLVESGGDLVKPGGSLRLSCAASGFTFSDYYMSWLRQAPGKELEWVSHISSSGTTTYYADSVEGRFTITRDNAKNSLYLQMNSLRAEDTAVYYCARGAAVAPGFDSRGQGTLVTVSS (SEQ ID NO: 25)

A suitable antibody may be selected from those provided herein fordevelopment and therapeutic or other use, including, without limitation,use as a multispecific antibody, such as a bispecific antibody.

Determination of affinity for a candidate protein can be performed usingmethods known in the art, such as Biacore measurements. Members of theantibody families described herein may have an affinity for IL2R with aKd of from about 10⁻⁶ to around about 10⁻¹, including withoutlimitation: from about 10⁻⁶ to around about 10⁻¹⁰; from about 10⁻⁶ toaround about 10⁻⁹; from about 10⁻⁶ to around about 10⁻⁸; from about 10⁻⁸to around about 10⁻¹¹; from about 10⁻⁸ to around about 10⁻¹⁰; from about10⁻⁸ to around about 10⁻⁹; from about 10⁻⁹ to around about 10⁻¹¹; fromabout 10⁻⁹ to around about 10⁻¹⁰; or any value within these ranges. Theaffinity selection may be confirmed with a biological assessment formodulating, e.g., agonizing, an IL2R biological activity, including invitro assays, pre-clinical models, and clinical trials, as well asassessment of potential toxicity.

Members of the antibody families described herein are cross-reactivewith the IL2R protein of Cynomolgus macaque, which facilitates the useof Cynomolgus macaque as an animal model for validating, e.g., mechanismof action, pharmacokinetics, toxicology, and other attributes of theantibodies described herein.

In some embodiments, the IL2R-specific antibodies herein comprise a VHdomain, comprising CDR1, CDR2, and CDR3 sequences in a human VHframework. The CDR sequences may be situated, as an example, in theregion of around amino acid residues 26-33; 51-58; and 97-116 for CDR1,CDR2, and CDR3, respectively, of the provided exemplary variable regionsequences set forth in SEQ ID NOs: 11-14 and 22-25. It will beunderstood by one of ordinary skill in the art that the CDR sequencesmay be in different positions if a different framework sequence isselected, although generally the order of the sequences will remain thesame.

In a particular embodiment, an anti-IL2RB antibody comprises a CDR1sequence of any one of SEQ ID NOs: 1-3. In a particular embodiment, theCDR1 sequence comprises SEQ ID NO: 1. In a particular embodiment, theCDR1 sequence comprises SEQ ID NO: 2. In a particular embodiment, theCDR1 sequence comprises SEQ ID NO: 3.

In a particular embodiment, an anti-IL2RB antibody comprises a CDR2sequence of any one of SEQ ID NOs: 4-6. In a particular embodiment, theCDR2 sequence comprises SEQ ID NO: 4. In a particular embodiment, theCDR2 sequence SEQ ID NO: 5. In a particular embodiment, the CDR2sequence comprises SEQ ID NO: 6.

In a particular embodiment, an anti-IL2RB antibody comprises a CDR3sequence of any one of SEQ ID NOs: 7-10. In a particular embodiment, theCDR3 sequence comprises SEQ ID NO: 7. In a particular embodiment, theCDR2 sequence comprises SEQ ID NO: 8. In a particular embodiment, theCDR2 sequence comprises SEQ ID NO: 9. In a particular embodiment, theCDR2 sequence comprises SEQ ID NO: 10.

In a further embodiment, an anti-IL2RB heavy chain-only antibodycomprises the CDR1 sequence of SEQ ID NO: 1; the CDR2 sequence of SEQ IDNO: 4; and the CDR3 sequence of SEQ ID NO: 7.

In a further embodiment, an anti-IL2RB antibody comprises the CDR1sequence of SEQ ID NO: 1; the CDR2 sequence of SEQ ID NO: 4; and theCDR3 sequence of SEQ ID NO: 8.

In a further embodiment, an anti-IL2RB antibody comprises the CDR1sequence of SEQ ID NO: 2; the CDR2 sequence of SEQ ID NO: 5; and theCDR3 sequence of SEQ ID NO: 9.

In a further embodiment, an anti-IL2RB antibody comprises the CDR1sequence of SEQ ID NO: 3, the CDR2 sequence of ID NO: 6; and the CDR3sequence of ID NO: 10.

In a further embodiment, an anti-IL2RB antibody comprises any of theheavy chain variable region amino acid sequences of SEQ ID NOs: 11-14(Table 2).

In a still further embodiment, an anti-IL2RB antibody comprises theheavy chain variable region sequence of SEQ ID NO: 11. In a stillfurther embodiment, an anti-IL2RB antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 12. In a still further embodimentan anti-IL2RB antibody comprises the heavy chain variable regionsequence of SEQ ID NO: 13. In a still further embodiment, an anti-IL2RBantibody comprises the heavy chain variable region sequence of SEQ IDNO: 14.

In some embodiments, a CDR sequence in an anti-IL2RB antibody of thedisclosure comprises one or two amino acid substitutions relative to aCDR1, CDR2, and/or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequencesin any one of SEQ ID NOs: 1-10 (Table 1).

In some embodiments, an anti-IL2RB antibody comprises a heavy chainvariable domain (VH) in which the CDR3 sequence has greater than orequal to 80%, such as at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity at the amino acid level to a CDR3 sequenceof any one of the antibodies whose CDR3 sequences are provided in Table1, and binds to IL2RB.

In some embodiments, an anti-IL2RB antibody comprises a heavy chainvariable domain (VH) in which the full set of CDRs 1, 2, and 3(combined) has greater than or equal to eighty-five percent (85%) (e.g.,≥90%, ≥95%, ≥98%, ≥99%) sequence identity at the amino acid level to theCDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences areprovided in Table 1, and binds to IL2RB.

In some embodiments, an anti-IL2RB antibody comprises a heavy chainvariable region sequence with at least about 80% identity, at least 85%identity, at least 90% identity, at least 95% identity, at least 98%identity, or at least 99% identity to any of the heavy chain variableregion sequences of SEQ ID NOs: 11-14 (shown in Table 2), and binds toIL2RB.

In some embodiments, an anti-IL2RB antibody comprises a heavy chainvariable region sequence as described herein, paired with a fixed lightchain sequence. In some embodiments, the fixed light chain comprises aCDR1 sequence of SEQ ID NO: 44, a CDR2 sequence of SEQ ID NO: 45, and aCDR3 sequence of SEQ ID NO: 46, in a human VL framework. Together, theanti-IL2RB VH region and the fixed light chain variable region havebinding affinity for IL2RB. In some embodiments, a fixed light chaincomprises a light chain variable region sequence of SEQ ID NO: 47. Insome embodiments, a fixed light chain comprises a sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 99% percent identity to the heavy chain variableregion sequence of SEQ ID NO: 47. In some embodiments, a fixed lightchain further comprises a light chain constant region sequence (CL). Insome embodiments, a fixed light chain comprises the sequence of SEQ IDNO: 48.

In some embodiments, an anti-IL2RB antibody is a heavy chain-onlyantibody comprising a heavy chain variable region sequence as describedherein, which is not paired with a light chain sequence.

In a particular embodiment, an anti-IL2RG antibody comprises a CDR1sequence of any one of SEQ ID NOs: 15-16. In a particular embodiment,the CDR1 sequence comprises SEQ ID NO: 15. In a particular embodiment,the CDR1 sequence comprises SEQ ID NO: 16.

In a particular embodiment, an anti-IL2RG antibody comprises a CDR2sequence of any one of SEQ ID NOs: 17-19. In a particular embodiment,the CDR2 sequence comprises SEQ ID NO: 17. In a particular embodiment,the CDR2 sequence comprises SEQ ID NO: 18. In a particular embodiment,the CDR2 sequence comprises SEQ ID NO: 19.

In a particular embodiment, an anti-IL2RG antibody comprises a CDR3sequence of any one of SEQ ID NOs: 20-21. In a particular embodiment,the CDR3 sequence comprises SEQ ID NO: 20. In a particular embodiment,the CDR2 sequence comprises SEQ ID NO: 21.

In a further embodiment, an anti-IL2RG heavy chain-only antibodycomprises the CDR1 sequence of SEQ ID NO: 15; the CDR2 sequence of SEQID NO: 17; and the CDR3 sequence of SEQ ID NO: 20.

In a further embodiment, an anti-IL2RG antibody comprises the CDR1sequence of SEQ ID NO: 15; the CDR2 sequence of SEQ ID NO: 18; and theCDR3 sequence of SEQ ID NO: 20.

In a further embodiment, an anti-IL2RG antibody comprises the CDR1sequence of SEQ ID NO: 16; the CDR2 sequence of SEQ ID NO: 18; and theCDR3 sequence of SEQ ID NO: 20.

In a further embodiment, an anti-IL2RG antibody comprises the CDR1sequence of SEQ ID NO: 15; the CDR2 sequence of SEQ ID NO: 19; and theCDR3 sequence of SEQ ID NO: 21.

In a further embodiment, an anti-IL2RG antibody comprises any of theheavy chain variable region amino acid sequences of SEQ ID NOs: 22-25(Table 4).

In a still further embodiment, an anti-IL2RG antibody comprises theheavy chain variable region sequence of SEQ ID NO: 22. In a stillfurther embodiment, an anti-IL2RB antibody comprises the heavy chainvariable region sequence of SEQ ID NO: 23. In a still furtherembodiment, an anti-IL2RB antibody comprises the heavy chain variableregion sequence of SEQ ID NO: 24. In a still further embodiment, ananti-IL2RB antibody comprises the heavy chain variable region sequenceof SEQ ID NO:25.

In some embodiments, a CDR sequence in an anti-IL2RG antibody of thedisclosure comprises one or two amino acid substitutions relative to aCDR1, CDR2 and/or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequencesin any one of SEQ ID NOs: 15-21 (Table 3).

In some embodiments, an anti-IL2RG antibody comprises a heavy chainvariable domain (VH) in which the CDR3 sequence has greater than orequal to 80%, such as at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity at the amino acid level to a CDR3 sequenceof any one of the antibodies whose CDR3 sequences are provided in Table3, and binds to IL2RG.

In some embodiments, an anti-IL2RG antibody comprises a heavy chainvariable domain (VH) in which the full set of CDRs 1, 2, and 3(combined) has greater than or equal to eighty-five percent (85%) (e.g.,≥90%, ≥95%, ≥98%, ≥99%) sequence identity at the amino acid level to theCDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences areprovided in Table 3, and binds to IL2RG.

In some embodiments, an anti-IL2RG antibody comprises a heavy chainvariable region sequence with at least about 80% identity, at least 85%identity, at least 90% identity, at least 95% identity, at least 98%identity, or at least 99% identity to any of the heavy chain variableregion sequences of SEQ ID NOs: 22-25 (shown in Table 4), and binds toIL2RG.

In some embodiments, an anti-IL2RG antibody comprises a heavy chainvariable region sequence as described herein, paired with a fixed lightchain sequence. In some embodiments, the fixed light chain comprises aCDR1 sequence of SEQ ID NO: 44, a CDR2 sequence of SEQ ID NO: 45, and aCDR3 sequence of SEQ ID NO: 46, in a human VL framework. Together, theanti-IL2RG VH region and the fixed light chain variable region havebinding affinity for IL2RG. In some embodiments, a fixed light chaincomprises a light chain variable region sequence of SEQ ID NO: 47. Insome embodiments, a fixed light chain comprises a sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 99% percent identity to the heavy chain variableregion sequence of SEQ ID NO: 47. In some embodiments, a fixed lightchain further comprises a light chain constant region sequence (CL). Insome embodiments, a fixed light chain comprises the sequence of SEQ IDNO: 48.

In some embodiments, an anti-IL2RG antibody is a heavy chain-onlyantibody comprising a heavy chain variable region sequence as describedherein, which is not paired with a light chain sequence.

Multispecific Antibodies

Aspects of the disclosure include multispecific, e.g., bispecific,antibodies, which may have any of the configurations discussed herein,including, without limitation, a bispecific, bivalent heavy-chainantibody comprising two non-identical heavy chain polypeptide subunitsthat are associated with one another via an asymmetric (e.g.,knobs-in-holes (KiH)) interface. In certain embodiments, a bispecific,bivalent heavy chain antibody can comprise two non-identical heavy chainpolypeptide subunits that are associated with one another via anasymmetric interface, and may optionally further include two identicalfixed light chain polypeptide subunits, each of which associates withone of the two heavy chain polypeptide subunits.

In some embodiments, a bispecific antibody comprises at least one heavychain variable region that binds to a first IL2R subunit, and at leastone heavy chain variable region that binds to a second IL2R subunit. Insome embodiments, a bispecific antibody comprises at least one heavychain variable region that binds to IL2RB, and at least one heavy chainvariable region that binds to IL2RG. In some embodiments, amultispecific antibody further comprises an Fc portion comprising CH2and/or CH3 and/or CH4 domains, in the absence of a CH1 domain.

Various formats of multispecific antibodies are within the ambit of thedisclosure, including, without limitation, single chain polypeptides,two chain polypeptides, three chain polypeptides, four chainpolypeptides, and multiples thereof. The multispecific antibodies hereinspecifically include those binding to IL2RB and IL2RG.

In some embodiments, a multispecific antibody comprises a first variableregion comprising a member of the IL2RB_F09 family, comprising a CDR1sequence comprising SEQ ID NO: 26, a CDR2 sequence comprising SEQ ID NO:27, and a CDR3 sequence comprising SEQ ID NO: 28, and a second variableregion comprising a member of the IL2RG_F16 family, comprising a CDR1sequence comprising SEQ ID NO: 32, a CDR2 sequence comprising SEQ ID NO:33, and a CDR3 sequence comprising SEQ ID NO: 20.

In some embodiments, a multispecific antibody comprises a first variableregion comprising a member of the IL2RB_F09 family, comprising a CDR1sequence comprising SEQ ID NO: 26, a CDR2 sequence comprising SEQ ID NO:27, and a CDR3 sequence comprising SEQ ID NO: 28, and a second variableregion comprising a member of the IL2RG_F18 family, comprising a CDR1sequence comprising SEQ ID NO: 15, a CDR2 sequence comprising SEQ ID NO:19, and a CDR3 sequence comprising SEQ ID NO: 21.

In some embodiments, a multispecific antibody comprises a first variableregion comprising a member of the IL2RB_F18 family, comprising a CDR1sequence comprising SEQ ID NO: 29, a CDR2 sequence comprising SEQ ID NO:30, and a CDR3 sequence comprising SEQ ID NO: 31, and a second variableregion comprising a member of the IL2RG_F16 family, comprising a CDR1sequence comprising SEQ ID NO: 32, a CDR2 sequence comprising SEQ ID NO:33, and a CDR3 sequence comprising SEQ ID NO: 20.

In some embodiments, a multispecific antibody comprises a first variableregion comprising a member of the IL2RB_F18 family comprising a CDR1sequence comprising SEQ ID NO: 29, a CDR2 sequence comprising SEQ ID NO:30, and a CDR3 sequence comprising SEQ ID NO: 31, and a second variableregion comprising a member of the IL2RG_F18 family, comprising a CDR1sequence comprising SEQ ID NO: 15, a CDR2 sequence comprising SEQ ID NO:19, and a CDR3 sequence comprising SEQ ID NO: 21.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1,a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 7,and a second variable region that binds to IL2RG, comprising a CDR1sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 17, and a CDR3sequence of SEQ ID NO: 20.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1,a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8,and a second variable region that binds to IL2RG, comprising a CDR1sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 18, and a CDR3sequence of SEQ ID NO: 20.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 2,a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 9,and a second variable region that binds to IL2RG, comprising a CDR1sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 18, and a CDR3sequence of SEQ ID NO: 20.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 3,a CDR2 sequence of SEQ ID NO: 6, and a CDR3 sequence of SEQ ID NO: 10,and a second variable region that binds to IL2RG, comprising a CDR1sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 17, and a CDR3sequence of SEQ ID NO: 20.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1,a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8,and a second variable region that binds to IL2RG, comprising a CDR1sequence of SEQ ID NO: 16, a CDR2 sequence of SEQ ID NO: 18, and a CDR3sequence of SEQ ID NO: 20.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1,a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8,and a second variable region that binds to IL2RG, comprising a CDR1sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 19, and a CDR3sequence of SEQ ID NO: 21.

Table 5 provides a summary of various CDR combinations of bispecificIL2RB×IL2RG antibodies in accordance with embodiments of the disclosure.

TABLE 5 Bispecific IL2RB x IL2RG Antibodies, CDR Sequence CombinationsFamily ID No. Combination CDR1 CDR2 CDR3 IL2RB_F09C GGSISSSDW IDHSGSTGRGSWELSDAFDI ** (SEQ ID NO: 1) (SEQ ID NO: 4) (SEQ ID NO: 7) IL2RG_F16AGFTFSDYY ISSSGDTI ARGDAVSITGDY (SEQ ID NO: 15) (SEQ ID NO: 17) (SEQ IDNO: 20) IL2RB_F09G GGSISSSDW IDHSGST ARGSWELTDAFDI ** (SEQ ID NO: 1)(SEQ ID NO: 4) (SEQ ID NO: 8) IL2RG_F16B GFTFSDYY ISSSGSTI ARGDAVSITGDY(SEQ ID NO: 15) (SEQ ID NO: 18) (SEQ ID NO: 20) IL2RB_F09K GGSISSSNWISHSGST GRGSWELTDAFDI ** (SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 9)IL2RG_F16B GFTFSDYY ISSSGSTI ARGDAVSITGDY (SEQ ID NO: 15) (SEQ ID NO:18) (SEQ ID NO: 20) IL2RB_F18E GFTFSSYG ISYDGSNK ARDLDYDVLTGDPVGGFDI **(SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO: 10) IL2RG_F16A GFTFSDYYISSSGDTI ARGDAVSITGDY (SEQ ID NO: 15) (SEQ ID NO: 17) (SEQ ID NO: 20)IL2RB_F09G GGSISSSDW IDHSGST ARGSWELTDAFDI ** (SEQ ID NO: 1) (SEQ ID NO:4) (SEQ ID NO: 8) IL2RG_F16C GFTFNDYY ISSSGSTI ARGDAVSITGDY (SEQ ID NO:16) (SEQ ID NO: 18) (SEQ ID NO: 20) IL2RB_F09G GGSISSSDW IDHSGSTARGSWELTDAFDI ** (SEQ ID NO: 1) (SEQ ID NO: 4) (SEQ ID NO: 8) IL2RG_F18AGFTFSDYY ISSSGTTT ARGAAVAPGFDS (SEQ ID NO: 15) (SEQ ID NO: 19) (SEQ IDNO: 21)

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a heavy chain variable regionsequence of SEQ ID NO: 11, and a second variable region that binds toIL2RG, comprising a heavy chain variable region sequence of SEQ ID NO:22.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a heavy chain variable regionsequence of SEQ ID NO: 12, and a second variable region that binds toIL2RG, comprising a heavy chain variable region sequence of SEQ ID NO:23.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a heavy chain variable regionsequence of SEQ ID NO: 13, and a second variable region that binds toIL2RG, comprising a heavy chain variable region sequence of SEQ ID NO:23.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a heavy chain variable regionsequence of SEQ ID NO: 14, and a second variable region that binds toIL2RG, comprising a heavy chain variable region sequence of SEQ ID NO:22.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a heavy chain variable regionsequence of SEQ ID NO: 12, and a second variable region that binds toIL2RG, comprising a heavy chain variable region sequence of SEQ ID NO:24.

In some embodiments, a bispecific antibody comprises a first variableregion that binds to IL2RB, comprising a heavy chain variable regionsequence of SEQ ID NO: 12, and a second variable region that binds toIL2RG, comprising a heavy chain variable region sequence of SEQ ID NO:25.

Table 6 provides a summary of various heavy chain variable regioncombinations of bispecific IL2RB×IL2RG antibodies in accordance withembodiments of the disclosure.

TABLE 6 Bispecific IL2RB x IL2RG antibodies, VH Sequence CombinationsFamily ID No. Combination VH Sequences IL2RB_F09CQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWIG **EIDHSGSTNYNPSLMSRVTISVDKSKNQFSLKLSSVTAADTAVYFCGRGS IL2RG_F16AWELSDAFDIRGQGTLVTVSS (SEQ ID NO: 11)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSSISSSGDTIYYADSVQGRFTLSRDNAENSLFLQMNSLRAEDTAVYYCARG DAVSITGDYRGQGTLVTVSS(SEQ ID NO: 22) IL2RB_F09GQVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVRQPPGKGLEWIG **EIDHSGSTNYNPSLMSRVTISVDKSKNQFSLKLSSVTAADTAVYFCARGS IL2RG_F16BWELTDAFDIRGQGTLVTVSS (SEQ ID NO: 12)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARG DAVSITGDYRGQGTLVTVSS(SEQ ID NO: 23) IL2RB_F09KQVQLQESSPGLVKPSETLSLTCTVSGGSISSSNWWSWVRQPPGKGLEWIG **EISHSGSTNYNPSLKSRVTISVDKSKNQFSLRLSSVTAADTAVYFCGRGSW IL2RG_F16BELTDAFDIRGQGTLVTVSS (SEQ ID NO: 13)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARG DAVSITGDYRGQGTLVTVSS(SEQ ID NO: 23) IL2RB_F18EQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREWV **AVISYDGSNKYYTDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYCA IL2RG_F16ARDLDYDVLTGDPVGGFDIWGQGTLVTVSS (SEQ ID NO: 14)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSSISSSGDTIYYADSVQGRFTLSRDNAENSLFLQMNSLRAEDTAVYYCARG DAVSITGDYRGQGTLVTVSS(SEQ ID NO: 22) IL2RB_F09GQVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVRQPPGKGLEWIG **EIDHSGSTNYNPSLMSRVTISVDKSKNQFSLKLSSVTAADTAVYFCARGS IL2RG_F16CWELTDAFDIRGQGTLVTVSS (SEQ ID NO: 12)QVQLVESGGGLVKPGGSLRLSCAASGFTFNDYYMSWIRQAPGKGLEWVSHISSSGSTIYYADSVKGRFTVSRDNANNSLYLQMHSLRAEDTAVYYCARG DAVSITGDYRGQGTLVTVSS(SEQ ID NO: 24) IL2RB_F09GQVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVRQPPGKGLEWIG **EIDHSGSTNYNPSLMSRVTISVDKSKNQFSLKLSSVTAADTAVYFCARGS IL2RG_F18AWELTDAFDIRGQGTLVTVSS (SEQ ID NO: 12)QVQLVESGGDLVKPGGSLRLSCAASGFTFSDYYMSWLRQAPGKELEWVSHISSSGTTTYYADSVEGRFTITRDNAKNSLYLQMNSLRAEDTAVYYCARG AAVAPGFDSRGQGTLVTVSS(SEQ ID NO: 25)

In some embodiments, a multispecific antibody includes a first and asecond polypeptide, i.e., a first and a second polypeptide subunit,wherein each polypeptide comprises an antigen-binding domain of aheavy-chain antibody. In some embodiments, each of the first and secondpolypeptides further includes a hinge region, or at least a portion of ahinge region, which can facilitate formation of at least one disulfidebond between the first and second polypeptides. In some embodiments,each of the first and second polypeptides further includes at least oneheavy chain constant region (CH) domain, such as a CH2 domain, and/or aCH3 domain, and/or a CH4 domain. In certain embodiments, the CH domainlacks a CH1 domain. The antigen-binding domain of each of the first andsecond polypeptides can incorporate any of the CDR sequences and/orvariable region sequences described herein in order to impartantigen-binding capability on the multispecific antibody. As such, incertain embodiments, each polypeptide subunit in a multispecificantibody can include an antigen-binding domain that binds to a differentIL2R subunit, or chain (e.g., IL2RB and IL2RG).

In some embodiments, a multispecific antibody comprises a variant humanIgG4 Fc domain comprising a first heavy chain constant region sequencecomprising an S228P mutation, an F234A mutation, an L235A mutation, anda T366W mutation (knob), and a second heavy chain constant regionsequence comprising an S228P mutation, an F234A mutation, an L235Amutation, a T366S mutation, an L368A mutation, and a Y407V mutation(hole). This variant, or modified, IgG4 Fc domain prevents unwanted Fabexchange, reduces effector function of the antibody, and alsofacilitates heterodimerization of the heavy chain polypeptide subunitsto form the multispecific (e.g., bispecific) antibody.

The components of the multispecific antibodies described herein (i.e.,CDR sequences, variable region sequences, and Fc domain sequences (e.g.,hinge, CH2, and CH3 domain sequences) can be combined in various ways togenerate multispecific antibodies that bind to IL2R, e.g., to IL2RB andIL2RG, and that have beneficial properties, e.g., reduced effectorfunction activity, increased IL2R agonistic activity, etc.

Table 7 provides the sequences of human IgG1 and IgG4 Fc regionsequences, as well as versions of these sequences that incorporateadditional mutations (variants) that impart additional desiredproperties.

TABLE 7 Human IgG1 and IgG4 Fc Region Sequences and Variants ThereofPolypeptide Name Amino Acid Sequence Human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS (UniProt No. P01857)WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 42)Human IgG4 ASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVS (UniProt No.P01861) WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 43) HumanIgG1 with ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL silencingmutations (Fc TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK region)VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO:51) Human IgG4 with ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALsilencing mutations (Fc TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTregion) KVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 52)Human IgG4 hinge ESKYGPPCPSCPA (SEQ ID NO: 54) region (wild type) HumanIgG4 hinge ESKYGPPCP P CPA (SEQ ID NO: 55) region (S228P) Human IgG4 CH2APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN domain sequence (wildWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY type) KCKVSNKGLPSSIEKTISKAK(SEQ ID NO: 56) Human IgG4 CH2 APE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN domain sequenceWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY (F234A, L235A)KCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 57) Human IgG4 CH3GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ domain sequence (wildPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA type) LHNHYTQKSLSLSLGK(SEQ ID NO: 58) Human IgG4 CH3 GQPREPQVYTLPPSQEEMTKNQVSL WCLVKGFYPSDIAVEWESNG domain sequenceQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE (knob, T366W)ALHNHYTQKSLSLSLGK (SEQ ID NO: 59) Human IgG4 CH3GQPREPQVYTLPPSQEEMTKNQVSL S C A VKGFYPSDIAVEWESNG domain sequence (hole,QPENNYKTTPPVLDSDGSFFL V SRLTVDKSRWQEGNVFSCSVMHE T366S, L368A,ALHNHYTQKSLSLSLGK (SEQ ID NO: 60) Y407V)

In some embodiments, a bispecific antibody comprises a first heavy chainpolypeptide subunit comprising a variable region that binds to IL2RB,wherein the first heavy chain polypeptide subunit comprises the sequenceof SEQ ID NO: 53, and a second heavy chain polypeptide subunitcomprising a variable region that binds to IL2RG, wherein the secondheavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 61.

In some embodiments, a bispecific antibody comprises a first heavy chainpolypeptide subunit comprising a variable region that binds to IL2RB,wherein the first heavy chain polypeptide subunit comprises the sequenceof SEQ ID NO: 62, and a second heavy chain polypeptide subunitcomprising a variable region that binds to IL2RG, wherein the secondheavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 63.

In some embodiments, a bispecific antibody comprises a first heavy chainpolypeptide subunit comprising a variable region that binds to IL2RB,wherein the first heavy chain polypeptide subunit comprises the sequenceof SEQ ID NO: 64, and a second heavy chain polypeptide subunitcomprising a variable region that binds to IL2RG, wherein the secondheavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 65.

In some embodiments, a bispecific antibody comprises a first heavy chainpolypeptide subunit comprising a variable region that binds to IL2RB,wherein the first heavy chain polypeptide subunit comprises the sequenceof SEQ ID NO: 66, and a second heavy chain polypeptide subunitcomprising a variable region that binds to IL2RG, wherein the secondheavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 67.

In some embodiments, a bispecific antibody comprises a first heavy chainpolypeptide subunit comprising a variable region that binds to IL2RB,wherein the first heavy chain polypeptide subunit comprises the sequenceof SEQ ID NO: 34, and a second heavy chain polypeptide subunitcomprising a variable region that binds to IL2RG, wherein the secondheavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 35.

In some embodiments, a bispecific antibody comprises a first heavy chainpolypeptide subunit comprising a variable region that binds to IL2RB,wherein the first heavy chain polypeptide subunit comprises the sequenceof SEQ ID NO: 36, and a second heavy chain polypeptide subunitcomprising a variable region that binds to IL2RG, wherein the secondheavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 37.

Table 8 provides a summary of various heavy chain polypeptide subunitsequence combinations of bispecific IL2RB×IL2RG antibodies in accordancewith embodiments of the disclosure.

TABLE 8 Bispecific IL2RB x IL2RG Antibodies, Full Length PolypeptideSequence Combinations Family ID No. Polypeptide Combination Name FullLength Sequences IL2RB_F09C hole IL2RB_F09CQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWV ** IgG4 holeRQPPGKGLEWIGEIDHSGSTNYNPSLMSRVTISVDKSKN IL2RG_F16AQFSLKLSSVTAADTAVYFCGRGSWELSDAFDIRGQGTL knobVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK(SEQ ID NO: 53) IL2RG_F16A QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIR IgG4knob QAPGKGLEWVSSISSSGDTIYYADSVQGRFTLSRDNAENSLFLQMNSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 61) IL2RB_F09Ghole IL2RB_F09G QVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVR ** IgG4 holeQPPGKGLEWIGEIDHSGSTNYNPSLMSRVTISVDKSKNQ IL2RG_F16BFSLKLSSVTAADTAVYFCARGSWELTDAFDIRGQGTLVT knobVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K(SEQ ID NO: 62) IL2RG_F16B QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIR IgG4knob QAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLW CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK(SEQ ID NO: 63) IL2RB_F09K hole IL2RB_F09KQVQLQESSPGLVKPSETLSLTCTVSGGSISSSNWWSWVR ** IgG4 holeQPPGKGLEWIGEISHSGSTNYNPSLKSRVTISVDKSKNQF IL2RG_F16BSLRLSSVTAADTAVYFCGRGSWELTDAFDIRGQGTLVT knobVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K(SEQ ID NO: 64) IL2RG_F16B QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIR IgG4knob QAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLW CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK(SEQ ID NO: 65) IL2RB_F18E hole IL2RB_F18EQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVR ** IgG4 holeQAPGKEREWVAVISYDGSNKYYTDSVKGRFTISRDNSK IL2RG_F16ANTLYLEMNSLRAEDTAVYYCARDLDYDVLTGDPVGGF knobDIWGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 66)IL2RG_F16A QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIR IgG4 knobQAPGKGLEWVSSISSSGDTIYYADSVQGRFTLSRDNAENSLFLQMNSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK(SEQ ID NO: 67) IL2RB_F09G hole IL2RB_F09GQVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVR ** IgG4 holeQPPGKGLEWIGEIDHSGSTNYNPSLMSRVTISVDKSKNQ IL2RG_F16CFSLKLSSVTAADTAVYFCARGSWELTDAFDIRGQGTLVT knobVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K(SEQ ID NO: 34) IL2RG_F16C QVQLVESGGGLVKPGGSLRLSCAASGFTFNDYYMSWIR IgG4knob QAPGKGLEWVSHISSSGSTIYYADSVKGRFTVSRDNANNSLYLQMHSLRAEDTAVYYCARGDAVSITGDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLW CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK(SEQ ID NO: 35) IL2RB_F09G hole IL2RB_F09GQVQLQESGPGLVKSSETLSLTCTVSGGSISSSDWWSWVR ** IgG4 holeQPPGKGLEWIGEIDHSGSTNYNPSLMSRVTISVDKSKNQ IL2RG_F18AFSLKLSSVTAADTAVYFCARGSWELTDAFDIRGQGTLVT knobVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K(SEQ ID NO: 36) IL2RG_F18A QVQLVESGGDLVKPGGSLRLSCAASGFTFSDYYMSWLR IgG4knob QAPGKELEWVSHISSSGTTTYYADSVEGRFTITRDNAKNSLYLQMNSLRAEDTAVYYCARGAAVAPGFDSRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK (SEQ ID NO: 37)

Additional sequences referred to herein are provided in Tables 9 and 10for reference.

TABLE 9 Additional Sequences UniProt KB Protein Name No. Amino AcidSequence Human IL2RA P01589 MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGC VFLLISVLLLSGLTWQRRQRKSRRTI(SEQ ID NO: 38) Human IL2RB P14784MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERV PRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGP REGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDP THLV (SEQ IDNO: 39) Human IL2RG P31785 MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKF SLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSN TSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 40) Human IL2 P60568MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO: 41)

TABLE 10 Additional Sequences Protein Name Amino Acid Sequence Fixedlight chain, QSVSSN (SEQ ID NO: 44) CDR1 sequence Fixed light chain, GAS(SEQ ID NO: 45) CDR2 sequence Fixed light chain, QQYNNWPWT (SEQ ID NO:46) CDR3 sequence Fixed light chain, VLEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLL sequenceIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP WTFGQGTKVEIK (SEQ IDNO: 47) Fixed light chain, fullEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLL length sequence (VL +IYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP CL)WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 48)Cellular Internalization

In some embodiments, the antibodies of the disclosure, once bound to abinding target (e.g., IL2R), internalize into cells, whereinternalization is at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, at leastabout 100%, at least about 110%, at least about 120%, at least about130%, at least about 140%, at least about 150%, at least about 160%, atleast about 170%, at least about 180%, at least about 190%, or at leastabout 200% or more, in comparison to one or more control antibodies thatdo not internalize. In some embodiments, aspects of the methodsdescribed herein involve internalizing an antibody as described hereinwithin a cell to achieve a desired effect, e.g., to act as an IL2Ragonist.

Cellular internalization results are provided in FIG. 7A and FIG. 7B.FIG. 7A depicts internalization of the indicated anti-IL2Rβ/γ UniAbs™ byCD8⁺ T-cells from human PBMCs, as a function of time. FIG. 7B depictsthis data in tabular format. Surface levels of UniAb™ were detected byflow cytometry and reported relative to cells which had not been allowedto internalize. The observed half-lives ranged from 0.27 hours to 0.81hours. As observed here, internalization was potentially partiallydependent on the specific anti-IL2RG arm of the bispecific antibody, asmolecules comprising the IL2RG_F16B binding sequence internalizedfaster, and to a greater degree, than molecules containing differentanti-IL2RG binding sequences.

Preparation of Anti-IL2R Antibodies

The antibodies of the present disclosure can be prepared by methodsknown in the art. In some embodiments, the antibodies herein areproduced by transgenic animals, including transgenic mice and rats,e.g., transgenic rats, in which the endogenous immunoglobulin genes areknocked out or disabled. In some embodiments, the heavy chain antibodiesherein are produced in a UniRat™ UniRat™ have their endogenousimmunoglobulin genes silenced and use a human immunoglobulin heavy-chaintranslocus to express a diverse, naturally optimized repertoire of fullyhuman HCAbs. While endogenous immunoglobulin loci in rats can be knockedout or silenced using a variety of technologies, in UniRat™ thezinc-finger (endo)nuclease (ZNF) technology was used to inactivate theendogenous rat heavy chain J-locus, light chain Cκ locus and light chainCλ locus. ZNF constructs for microinjection into oocytes can produce IgHand IgL knock out (KO) lines. For details, see, e.g., Geurts et al.,2009, Science 325:433. Characterization of Ig heavy chain knockout ratshas been reported by Menoret et al., 2010, Eur. J. Immunol.40:2932-2941. Advantages of the ZNF technology are that non-homologousend joining to silence a gene or locus via deletions up to several kbcan also provide a target site for homologous integration (Cui et al.,2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced inUniRat™ are called UniAbs™ and can bind epitopes that cannot be attackedwith conventional antibodies. Their high specificity, affinity, andsmall size make them ideal for mono- and poly-specific applications.

In addition to UniAbs™, specifically included herein are heavychain-only antibodies lacking the camelid VHH framework and mutations,and their functional VH regions. Such heavy chain-only antibodies can,for example, be produced in transgenic rats or mice which comprise fullyhuman heavy chain-only gene loci as described, e.g., in WO2006/008548,but other transgenic mammals, such as rabbit, guinea pig, and rat canalso be used. Heavy chain-only antibodies, including their VHH or VHfunctional fragments, can also be produced by recombinant DNAtechnology, by expression of the encoding nucleic acid in a suitableeukaryotic or prokaryotic host, including, for example, mammalian cells(e.g., CHO cells), E. coli, or yeast.

Domains of heavy chain-only antibodies combine advantages of antibodiesand small molecule drugs: can be mono- or multi-valent; have lowtoxicity; and are cost-effective to manufacture. Due to their smallsize, these domains are easy to administer, including oral or topicaladministration, are characterized by high stability, includinggastrointestinal stability; and their half-life can be tailored to thedesired use or indication. In addition, VH and VHH domains of HCAbs canbe manufactured in a cost-effective manner.

In a particular embodiment, the heavy chain antibodies of the presentdisclosure, including UniAbs™, have the native amino acid residue at thefirst position of the FR4 region (amino acid position 101 according tothe Kabat numbering system), substituted by another amino acid residue,which is capable of disrupting a surface-exposed hydrophobic patchcomprising or associated with the native amino acid residue at thatposition. Such hydrophobic patches are normally buried in the interfacewith the antibody light chain constant region but become surface exposedin HCAbs and are, at least partially, for the unwanted aggregation andlight chain association of HCAbs. In some embodiments, the substitutedamino acid residue is charged. In some embodiments, the substitutedamino acid residue is positively charged, such as lysine (Lys, K),arginine (Arg, R) or histidine (His, H), e.g., arginine (R). In someembodiments, the heavy chain-only antibodies derived from the transgenicanimals contain a Trp to Arg mutation at position 101. In someembodiments, the resultant HCAbs have high antigen-binding affinity andsolubility under physiological conditions in the absence of aggregation.

As part of the present disclosure, human IgG anti-IL2R heavy chainantibodies with unique sequences from UniRat™ animals (UniAb™) wereidentified that bind to human IL2R in ELISA protein and cell-bindingassays. The identified heavy chain variable region (VH) sequences arepositive for human IL2R protein binding and/or for binding to IL2R+cells, and are all negative for binding to cells that do not expressIL2R.

Heavy chain antibodies binding to non-overlapping epitopes on an IL2Rprotein, e.g., UniAbs™ can be identified by competition binding assays,such as enzyme-linked immunoassays (ELISA assays) or flow cytometriccompetitive binding assays. For example, one can use competition betweenknown antibodies binding to the target antigen and the antibody ofinterest. By using this approach, one can divide a set of antibodiesinto those that compete with the reference antibody and those that donot. The non-competing antibodies are identified as binding to adistinct epitope that does not overlap with the epitope bound by thereference antibody. Often, one antibody is immobilized, the antigen isbound, and a second, labeled (e.g., biotinylated) antibody is tested inan ELISA assay for ability to bind the captured antigen. This can beperformed also by using surface plasmon resonance (SPR) platforms,including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GEHealthcare Life Sciences), and MX96 SPR imager (Ibis technologies B.V.),as well as on biolayer interferometry platforms, such as Octet Red384and Octet HTX (ForteBio, Pall Inc). For further details, see theExamples herein.

Typically, an antibody “competes” with a reference antibody if it causesabout 15-100% reduction in the binding of the reference antibody to thetarget antigen, as determined by standard techniques, such as by thecompetition binding assays described above. In some embodiments,competitive binding is measured using an enzyme-linked immunoassay(ELISA assay). In some embodiments, one antibody is immobilized, theantigen is bound, and a second, labeled (e.g., biotinylated) antibody istested in an ELISA assay for ability to bind the captured antigen. Thiscan be performed, for example, using a surface plasmon resonance (SPR)platform, such as, for example, ProteOn XPR36 (BioRad, Inc), Biacore2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR imager(Ibis technologies B.V.), as well as on biolayer interferometryplatforms, such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). Insome embodiments, competitive binding is measured using a flowcytometric competitive binding assay.

In various embodiments, the relative inhibition is at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50% atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% or higher.

Pharmaceutical Compositions

It is another aspect of the present disclosure to provide pharmaceuticalcompositions comprising one or more antibodies of the present disclosurein admixture with a suitable pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers as used herein are exemplified, butnot limited to, adjuvants, solid carriers, water, buffers, or othercarriers used in the art to hold therapeutic components, or combinationsthereof.

In one embodiment, a pharmaceutical composition comprises a heavy chainantibody (e.g., UniAb™) that binds to IL2R. In another embodiment, apharmaceutical composition comprises a multi-specific (includingbispecific) heavy chain antibody (e.g., UniAb™) that binds to two ormore non-overlapping epitopes on an IL2R protein (e.g., a first epitopeon a first IL2R polypeptide chain (e.g., IL2RB) and a second epitope ona second IL2R polypeptide chain (e.g., IL2RG). In some embodiments, apharmaceutical composition comprises a multi-specific (includingbispecific) heavy chain antibody (e.g., UniAb™) that binds to IL2RB andIL2RG.

Pharmaceutical compositions of the antibodies used in accordance withthe present disclosure are prepared for storage by mixing proteinshaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (see, e.g. Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as inthe form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

In some embodiments, pharmaceutical compositions for parenteraladministration are sterile and substantially isotonic and manufacturedunder Good Manufacturing Practice (GMP) conditions. Pharmaceuticalcompositions can be provided in unit dosage form (i.e., the dosage for asingle administration). The formulation depends on the route ofadministration chosen. The antibodies herein can be administered byintravenous injection or infusion or subcutaneously. For injectionadministration, the antibodies herein can be formulated in aqueoussolutions, e.g., in physiologically-compatible buffers to reducediscomfort at the site of injection. The solution can contain carriers,excipients, or stabilizers as discussed above. Alternatively, antibodiescan be in lyophilized form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

Antibody formulations are disclosed, for example, in U.S. Pat. No.9,034,324. Similar formulations can be used for the heavy chainantibodies, including UniAbs™, of the present disclosure. Subcutaneousantibody formulations are described, for example, in US20160355591 andUS20160166689.

Methods of Use

The anti-IL2R antibodies and pharmaceutical compositions describedherein can be used for the treatment of diseases and conditions that aremediated by activation of IL2R signaling in immune cells, such as immuneeffector cells, such as effector T and natural killer (NK) cells. Insome embodiments, the disease or condition can be an infectious disease,an autoimmune disorder (e.g., Crohn's disease, Multiple Sclerosis), acancer, an inflammatory disease (e.g., arthritis), a disease or disorderassociated with deficient IL-2-mediated signaling, deficient T cellproliferation, or T cell dysfunction.

In some embodiments, a disease or disorder is one in which increasedIL-2-mediated signaling is therapeutic to the patient. In someembodiments, a disease or disorder is associated with a deficient T cellresponse, e.g., a deficient CD8+ T cell response.

In some embodiments, a treatment is aimed at preventing or treating adisease or disorder by: increasing the number of CD3+ T cells,increasing the number of CD4+ T cells, increasing the number of CD8+ Tcells, increasing the number of CD8+ effector T cells (e.g., CTLs),increasing the number NK cells, increasing the ratio of CD8+ T cells toCD4+ T cells, decreasing the proportion of Tregs, or any combinationthereof.

In some embodiments, a disease or disorder can manifest as an infection,or as an inability to mount an effective immune response against aninfection. The infection may be chronic, persistent, latent, or slow,and may be the result of bacterial, viral, fungal, or parasiticinfection. As such, treatment may be provided to patients having abacterial, viral, or fungal infection. Non-limiting examples ofbacterial infections include infection with Helicobacter pylori.Non-limiting examples of viral infections include infection with EBV,HIV, hepatitis B or hepatitis C.

In some embodiments, a disease or disorder can be associated with acancer, such as, e.g., tumor immune escape. Many human tumors expresstumor-associated antigens recognised by T cells and capable of inducingan immune response. Cancers may also be treated where there is noindication of a T-cell dysfunctional disorder, but the use of anantibody described herein promotes an effective immune response.

In some embodiments, a treatment is aimed at prevention of a disease ordisorder associated with deficient and/or reduced IL-2-mediatedsignaling. As such, the antibodies described herein can be used toformulate pharmaceutical compositions or medicaments, and subjects maybe prophylactically treated against development of a disease state. Thismay take place before the onset of symptoms of the disease state, and/orsuch treatment can be given to subjects considered to be at greater riskof the disease or disorder.

In certain embodiments, the methods herein involve inducing activationof immune effector cells without preferentially activating regulatoryT-cells (Tregs). Without being held to theory, the inventors havediscovered that multispecific antibodies that simultaneously target boththe beta and gamma subunits of the IL2 receptor to induce activation(i.e., act as agonists) of IL2R signaling in human immune effector cellswithout preferentially activating Tregs, shifting the balance to theactivation of T-effector and NK cells, lead to improved treatmentoutcomes in diseases and disorders that are mediated by activation ofIL2R signaling.

Accordingly, aspects of the disclosure include methods of treatmentwherein a subject's immune response is boosted or aided by administeringa therapeutically effective amount of the one or more of the antibodiesdescribed herein. In some embodiments, the methods involve administeringan antibody described herein to achieve an immune response that destroyscancerous cells. In certain embodiments, the antibodies described hereinact as agonists of the IL2R signaling pathway to achieve such results.Methods in accordance with embodiments of the disclosure also includecombination therapy, wherein a subject is administered an antibody asdescribed herein, in conjunction with another course of therapy, e.g., achemotherapy regimen.

In one aspect, a multispecific (e.g., bispecific) antibody as describedherein, having agonist activity for the IL2R signaling pathway, is usedfor treating cancer. Cancers that are amenable to such treatmentinclude, but are not limited to, advanced or metastatic cancers. In someembodiments, a cancer is a solid tumor cancer. Solid tumor cancers inaccordance with embodiments of the disclosure include, but are notlimited to, renal cell carcinoma, melanoma, urothelial cancer, triplenegative breast cancer, non-small cell lung cancer (NSCLC), colorectalcancer, sarcoma, squamous cell carcinoma of the head and neck, andmetastatic castration-resistant prostate cancer.

Aspects of the disclosure also include methods for stimulating IL2Rsignaling in an immune cell, wherein the methods involve contacting theimmune cell with an antibody as described herein (e.g., with anagonistic bispecific antibody as described herein). In certainembodiments, the methods involve stimulating an IL2RB/IL2RG dimericreceptor complex on an immune cell by contacting the immune cell with amultispecific (e.g., bispecific) antibody that binds to both IL2RB andIL2RG, and acts as an agonist of the IL2R complex. Any of a variety ofimmune cells that express IL2R can be involved in the subject methods,including, but not limited to: CD4+ T-cells, CD8+ T-cells, and NaturalKiller (NK) cells.

Effective doses of the compositions of the present disclosure for thetreatment of disease vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g., companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

Dosage levels can be readily determined by the ordinarily skilledclinician, and can be modified as required, e.g., as required to modifya subject's response to therapy. The amount of active ingredient thatcan be combined with the carrier materials to produce a single dosageform varies depending upon the host treated and the particular mode ofadministration. Dosage unit forms generally contain between from about 1mg to about 500 mg of an active ingredient.

In some embodiments, the therapeutic dosage the agent may range fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once every two weeks or once a month oronce every 3 to 6 months. Therapeutic entities of the present disclosureare usually administered on multiple occasions. Intervals between singledosages can be weekly, monthly, or yearly. Intervals can also beirregular as indicated by measuring blood levels of the therapeuticentity in the patient. Alternatively, therapeutic entities of thepresent disclosure can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the polypeptidein the patient.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The pharmaceutical compositions herein are suitable for intravenous orsubcutaneous administration, directly or after reconstitution of solid(e.g., lyophilized) compositions. The preparation also can be emulsifiedor encapsulated in liposomes or micro particles such as polylactide,polyglycolide, or copolymer for enhanced adjuvant effect, as discussedabove. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug DeliveryReviews 28: 97-119, 1997. The agents of this disclosure can beadministered in the form of a depot injection or implant preparationwhich can be formulated in such a manner as to permit a sustained orpulsatile release of the active ingredient. The pharmaceuticalcompositions are generally formulated as sterile, substantially isotonicand in full compliance with all Good Manufacturing Practice (GMP)regulations of the U.S. Food and Drug Administration.

Toxicity of the antibodies and antibody structures described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD50 (the dose lethal to50% of the population) or the LD100 (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is thetherapeutic index. The data obtained from these cell culture assays andanimal studies can be used in formulating a dosage range that is nottoxic for use in humans. In some embodiments, the dosage of theantibodies described herein lies within a range of circulatingconcentrations that include the effective dose with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration, and dosage can be chosen by theindividual physician in view of the patient's condition.

The compositions for administration will commonly comprise an antibodyor other ablative agent dissolved in a pharmaceutically acceptablecarrier, e.g., an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well-known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, and thelike, e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of active agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs (e.g., Remington's Pharmaceutical Science (15th ed.,1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics(Hardman et al., eds., 1996)).

Also within the scope of the disclosure are kits comprising the activeagents and formulations thereof, of the disclosure and instructions foruse. The kit can further contain a least one additional reagent, e.g., achemotherapeutic drug, etc. Kits typically include a label indicatingthe intended use of the contents of the kit. The term “label” as usedherein includes any writing, or recorded material supplied on or with akit, or which otherwise accompanies a kit

Example embodiments now being fully described, it will be apparent toone of ordinary skill in the art that various changes and modificationscan be made without departing from the spirit or scope of thedisclosure.

EXAMPLES

Materials and Methods:

Antibody Construct Names

The following table (Table 11) provides shorthand nomenclature for sixbispecific antibody constructs evaluated herein:

TABLE 11 Shorthand Nomenclature Shorthand Name Sequence Name BsAb-1IL2RB_F09C ** IL2RG_F16A BsAb-2 IL2RB_F09G ** IL2RG_F16B BsAb-3IL2RB_F09G ** IL2RG_F16C BsAb-4 IL2RB_F09G ** IL2RG_F18A BsAb-5IL2RB_F09K ** IL2RG_F16B BsAb-6 IL2RB_F18E ** IL2RG_F16AImmunizations, Next-Generation Sequencing, Clonotype Analysis andCloning

Methods essentially as described in Harris et al. Front Immunol. 2018Apr. 24; 9:889(60). In brief, UniRat animals were immunized usingstandard adjuvants (Complete Freunds or Titermax/Ribi) along withrecombinant protein antigens in a 48-day protocol or DNA immunizations.For protein immunizations, boosts consisted of 10 μg of recombinantprotein injected into each leg of each animal with the appropriateadjuvant. In the case of DNA immunizations, gold particles were coatedwith vectors containing cDNA of the target antigen, which weresubsequently administered subcutaneously every 7 days, using a gene gun.Plasma samples were collected post-immunization to assess serum titersagainst the antigen by ELISA.

After approximately 7 weeks (protein antigen) or 10 weeks (DNA antigen)of immunization, draining lymph nodes were harvested and total RNA wasisolated. Ig heavy chain sequences were amplified using first strandcDNA synthesis and 5′ RACE by PCR, following methods similar to thosepreviously described in Harris et al. Front Immunol. 2018 Apr. 24; 9:889and then purified by gel extraction.

Next-generation sequencing was completed using the MiSeq platform(Illumina) with 2×300 paired-end reads. To enable multiplexing ofsamples, indexing labels were added by primer extension. Approximately100,000 paired reads covered each sample, and those that showedalignment of less than 20 nucleotides to a human Ig locus werediscarded. Merged forward and reverse reads of VH regions weretranslated into open reading frames and framework and CDR regionsidentified by IGBLAST (https://www.ncbi.nlm.nih.gov/igblast/).Clonotypes (defined by CDR3 protein sequences with at least 80% sequencesimilarity) were determined for samples using agglomerative clustering.CDR3 clonotypes were ranked by the percent of total reads in a sampledefined by that clonotype. Those with the greatest abundance wereprioritized for high-throughput cloning into an expression vectorcontaining a CH1-deleted human IgG1 Fc region and validated by Sangersequencing. Plasmids were transformed into E. coli grown in LB culturemedia and then purified to enable transient transfection of HEK 293cells in 96-well format. Following several days of expression,supernatants containing antibody were harvested and clarified bycentrifugation.

High-Throughput ELISA

Methods are essentially as described in Harris et al. Front Immunol.2018 Apr. 24; 9:889. Briefly, recombinant proteins were coated overnightat 4° C. in 96-well plates using BupH Carbonate-Bicarbonate buffer(human IL-2Rβ, Acrobiosystems; cynomolgus IL-2Rβ, Sino Biological).Plates were then washed with TBST (20 mM Tris, 150 mM NaCl, 0.05%Tween-20, pH 7.6) and blocked with blocking buffer (TBST with 1% drymilk powder). HEK 293 supernatants containing antibodies were diluted1:100 in blocking buffer and added to antigen-coated plates. Detectionof bound antibodies was accomplished using an HRP-labeled anti-human Igsecondary antibody together with chemiluminescent substrate.

Luminescence was quantified (SpectraMax i3X, Molecular devices) and thesignal for each well was normalized by dividing by the averagebackground luminescence of antigen-coated wells that had been incubatedwith supernatant from untransfected HEK 293 cells.

Cell Lines and PBMCs

M07e cells were obtained from DSMZ and were grown in RPMI mediumcontaining 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, and10 ng/mL rhGM-CSF. HSC-F cells were obtained from The Nonhuman PrimateReagent Resource and cultured in RPMI medium supplemented with 20% FBS,1% Penicillin/Streptomycin and 55 μM β-Mercaptoethanol. 293-F wereobtained from Gibco and grown according to their recommendations.

For creating stable cell lines expressing human IL-2Rβ or cynomolgusIL-2Rβ, expression constructs carried the full-length cDNA for theantigen and a NeoR selection cassette. Each expression construct wasthen linearized and used to electroporate CHO cells. Three days aftertransfection, cells were put under selection for 3-6 weeks usingGeneticin treatments. At the end of the selection period, alluntransfected and negative control cell lines were killed, while alltransfected pools showed regrowth as expected for successfullytransfected pools. Four pools of each target were then assayed by flowcytometry for binding to a positive control antibody. The culture mediafor the CHO cells was EX-CELL® 325 PF CHO media containing 8 mML-glutamine, 0.1 μg/L IGF-1, 5% dialyzed FBS, 0.45 mg/mL geneticin, and0.45 mg/mL hygromycin. The cells were grown in suspension and maintainedat a concentration between 0.5×10⁶/mL to 2×10⁶/mL.

Human PBMCs were isolated in-house from fresh leukapheresis packs(StemCell) by Ficoll® Paque Premium (GE Healthcare Life Sciences)density gradient centrifugation.

Cell Binding by Flow Cytometry

All washes and dilutions of cells, antibodies, and reagents wereperformed using flow buffer (1×PBS, 1% BSA, 0.1% NaN3, pH 7.4). Stainingwas performed in a round-bottom 96-well plate (Corning) seeded at100,000 cells/well and all incubations were performed at 4° C. or onice. For primary and secondary screens, the cells were incubated for 30minutes with pre-diluted test antibodies (secondary screen anddose-curves) or 1:5 diluted HEK 293 supernatants containing antibodies(for primary screens and diversity screens) in a total volume of 50 μL.The cells were washed twice with 200 μL flow buffer. The cells were thenincubated for 30 minutes with detection antibody (Goat F(ab′)2Anti-Human IgG-PE, Southern Biotech) at 0.625 μg/mL in flow buffer.Following 2 more washes, the cells were resuspended in a final volume of150 μL of flow buffer. The cells were analyzed on a BD FACSCelesta or aGuava easyCyte 8-HT flow cytometer. At least 3000 events were collected,and PE geometric mean fluorescence intensity was plotted as a fold overbackground (cells incubated with secondary detection antibody only). Insome secondary screens involving human or cynomolgus PBMCs, anadditional CD4 antibody (BioLegend) and/or CD8 antibody (BioLegend) wasincluded to further characterize cell binding.

pSTAT5 Detection by Flow Cytometry

For detection of pSTAT5 by flow cytometry, PBMCs were prepared fromeither frozen whole blood (cynomolgus) or frozen LeukoPak (human). Cellswere thawed, washed twice with complete RPMI medium and resuspended at5×10⁶ cells/mL. 100 μL/well of these cells was then transferred to asterile, round-bottom, 96-well plate (Corning) and sealed with anAeraSeal™ (Excel Scientific). The plate was then incubated at 37° C. and5% CO₂ for 1 hour. After the incubation, 100 μL of pre-dilutedantibodies (or IL-2/IL-2 variant) was added to the appropriate wells. Afinal concentration of 10 nM IL-2 (R&D Systems) was used in controlwells to ensure detectable pSTAT5. The plate was then resealed andreturned to the incubator for an additional 1 hour. After theincubation, the cells were centrifuged and washed twice with PBSpre-chilled to 4° C. The cells were then blocked with Human TruStain FcX(BioLegend) and then subsequently stained for 30 minutes with FixableViability Dye (Invitrogen) and antibodies against CD3, CD4, CD8, CD25,and/or CD56. After staining, the cells were again centrifuged and washedtwice with pre-chilled PBS. The cells were then fixed with the additionof 200 μL/well Fixation Buffer (BioLegend) and incubated at room tempfor 30 minutes. After fixation, the cells were centrifuged and washedtwice with Flow Buffer (1×PBS, 1% BSA, 0.1% NaN₃, pH 7.4). Next, thecells were permeabilized by resuspending in 200 μL/well True-Phos buffer(BioLegend) pre-chilled to −20° C. and transferred to a −20° C. freezerovernight. The following morning, the cells were centrifuged, washedtwice with flow buffer, and subsequently stained for 30 minutes withanti-pSTAT5 (BD Biosciences). After two additional washes, the cellswere resuspended in 125 μL/well flow buffer and acquired on a BDFACSCelesta.

Ki67 Detection by Flow Cytometry

For detection of Ki67 by flow cytometry, frozen human PBMCs (previouslyisolated in-house from a LeukoPak) were thawed and rested overnight incomplete RPMI medium at 1×10⁶ cells/mL. The morning of the assay, thePBMCs were washed with complete RPMI and resuspended at 1e6 cells/mL.Then, to each well of a sterile 96-well plate, 100 μL of PBMCs, 50 μL of0.16× ImmunoCult (StemCellTech), and 50 μL of diluted antibody or rhIL-2(R&D Systems) was added. 0.5× ImmunoCult was used for staining controlsto ensure detectable Ki67 and CD25 signal for compensation. The platewas then covered and incubated at 37° C. and 5% CO₂. After 3 days, themedia was refreshed with 100 μL/well of the corresponding concentrationantibody and ImmunoCult and then returned to the incubator. After 3 moredays (6 days total), the cells were centrifuged and washed twice withPBS pre-chilled to 4° C. The cells were then blocked with Human TruStainFcX (BioLegend) and then subsequently stained for 30 minutes withFixable Viability Dye (Invitrogen) and antibodies against CD3, CD4, CD8,CD25, and/or CD56. After staining, the cells were again centrifuged andwashed twice with pre-chilled PBS. The cells were then fixed andpermeabilized for 1 hour with 200 μL/well FoxP3/Transcription FactorStaining Buffer working solution (Invitrogen). After permeabilization,the cells were centrifuged, washed twice with permeabilization buffer,and subsequently stained for 30 minutes with anti-FoxP3 (BioLegend) andanti-Ki67 (BioLegend). After two additional washes, the cells wereresuspended in 125 μL/well flow buffer and acquired on a BD FACSCelesta.

Whole Blood Cytokine Release Assay

Cytokine secretion was detected using fresh human whole blood(heparinized) obtained from AllCells. The following method was adaptedfrom B. Wolf et al. Cytokine 60 (2012) 828-837(61). 12.5 μL of 20×concentrated (diluted in 1×PBS) test article was added to each well of asterile 96-well round bottom plate. To this, 237.5 μL of fresh, humanwhole blood was added to each well with minimal pipetting to reducenon-specific activation. The plate was the covered and incubated at 37°C. and 5% CO₂ overnight. The following morning, the plate wascentrifugated at 1800×g for 10 minutes and then 50 μL of serum wastransferred to a 96-well microplate. The serum was then immediatelytested by MSD (#K15010K-1 or a custom U-Plex plate) or frozen at −80° C.for later testing.

Mouse Pharmacokinetic (PK) Evaluation

The PK of BsAb-1 and BsAb-2 were each evaluated in 6 male BALB/c micefollowing a single tail vein injection of 1 mg/kg (n=3*6 groups, AragenBiosciences, Morgan Hill, CA). Serum samples were collected at selectedtime points over the course of 14 days post-dose.

The PK of BsAb-5 was evaluated in two groups of nine female BALB/c micefollowing a single tail vein injection of 1 mg/kg or 10 mg/kg (9 miceper dosing group, CrownBio, San Diego, CA). Serum samples were collectedat selected time points over the course of 14 days post-dose.

Mouse Accelerated GVHD Study

Each immune-compromised NSG mouse (8-9 weeks old from Charles River,France) was irradiated with 1.5 Gy on study day −1. Mice were dividedinto 4 groups (n=5) and 2 independent experiments were conducted using 2different PBMC donors. On study day 0, each mouse was adoptivelytransferred IV with 20 million human PBMCs from one of the 2 donors andeach mouse was treated with either vehicle control (100 μL), 22 μgrhIL-2 (350,000 UI/mice, Proleukin, Novartis) daily, 1 mg/kg BsAb-1twice a week or 1 mg/kg BsAb-2 twice a week. GVHD was assessed bymeasuring weight loss over time in all animals. Animals were euthanizedwhen body weight loss of 20% was observed.

In the second experiment, NSG mice (8-9 weeks old from Charles River,France) were irradiated with 1.5 Gy on study day −1. Mice were dividedinto 4 groups and 2 independent experiments were conducted using 2different PBMC donors. On study day 0, each mouse was adoptivelytransferred IV with 20 million CSFE-labelled human PBMCs from one of the2 donors and each mouse was treated with either vehicle control (100 μL)(n=7), 22 μg rhIL-2 (350,000 UI/mice) daily (n=6), 1 mg/kg BsAb-1 twicea week (n=6) or 1 mg/kg BsAb-2 twice a week (n=6). All animals weresacrificed on study day 5.

Immunophenotyping of the engrafted PBMCs by flow cytometry was performedon single-cell suspensions prepared from the mouse spleen on the day ofsacrifice. The method of detection was largely the same as the abovemethod for Ki67 detection by flow cytometry, but with different panelsof antibodies to better distinguish the human PBMCs from the host cells.Cells were surface stained with anti-human CD45, CD3, CD4, CD8, CD25,CD16, CD19, and/or CD69. Following fixation and permeabilization, someof the cells were stained with anti-human FoxP3. The samples were thencollected on a flow cytometer and analyzed using FlowJo analysissoftware.

For BsAb-5, 10 million PBMCs were transferred instead of 20 million, andmice were divided into 3 groups.

Cynomolgus Pharmacodynamic (PD) Study

The PD profiles of BsAb-1 and BsAb-2 were evaluated in twelve 2-4 yearsold naïve cynomolgus monkeys following a single IV (slow bolus) dose of0.03, 0.1 or 0.3 mg/kg. Each treatment group contained 1 male and 1female cynomolgus monkey (Charles River Lab, USA, Reno, NV). Bloodsamples were collected at selected time points for 21 days after dosingfor analyses of hematology, serum chemistry, cytokines, and PDendpoints. After study termination, animals from the study were returnedto the general colony. All procedures were approved by CRL IACUC andwere performed in compliance with the Animal Welfare Act, the Guide forCare and Use of Laboratory Animals and the Office of Laboratory AnimalWelfare.

Cynomolgus Blood Immunophenotyping

A portion of the blood from each collected time point was used forimmunophenotyping and quantification by flow cytometry. The method forKi67 detection by flow cytometry was largely the same as describedabove, but with a different panel of cyno-reactive antibodies. Cellswere surface stained with antibodies against CD3, CD4, CD8, CD20, CD25,and CD159a. After fixation and permeabilization, the cells were stainedwith antibodies against FoxP3 and Ki67. The samples were then collectedon a flow cytometer and analyzed using FlowJo analysis software.

Simultaneously, a portion of each blood sample was transferred to BDTruCount tubes and stained with CD45 for real time quantification ofperipheral blood cell absolute counts. The cell subset percentages fromthe above blood analysis were applied to the total cell numbers from thecorresponding TruCount tube.

αIgG4 ELISA

Serum concentrations of BsAb-1 and BsAb-2 in mouse serum were determinedusing an antigen capture ELISA. All washes and dilutions were performedwith freshly made TBS-T (Accuris). All volumes should be assumed to be100 μL/well except for coating, blocking and washing, which are at 200μL/well. The night before the assay, Nunc MaxiSorp™ flat-bottom plates(Invitrogen) were coated with recombinant human IL2Rγ protein diluted to1 μg/mL in carbonate-bicarbonate buffer (Thermo Scientific) and left at4° C. The next day, the plates were washed 5 times and then blocked with1% BSA for 30 minutes. The plates were washed once and then multipledilutions of the serum samples were added, along with a referencestandard. Stocks of known concentration for BsAb-1 and BsAb-2 were usedto make the standard curve. After 1 hour at room temp, the plate waswashed 8 times and then biotinylated anti-human IgG4-Fc (MABTECH)diluted to 3 μg/mL was added. The plates were incubated at room temp foranother 30 minutes and then washed again 8 times. Next, the plates wereincubated for 30 minutes with HRP-Streptavidin (Thermo Scientific)diluted 1:4000. Following an additional 8 washes, the plates wereincubated in the dark for 6 minutes with room-temperature 1-Step UltraTMB (Thermo Scientific). The reaction was stopped with 100 μL/well 2 Nsulfuric acid. Absorbance was assessed at 450 nm and 570 nm.

Protein Expression and Purification

Monospecific UniAbs were expressed in ExpiCHO cells following themanufacturer's instructions (ThermoFisher A29133, Standard Protocol).Clarified supernatants were harvested on day 7 and purified usingProtein A magnetic beads, using the KingFisher Flex Platform(ThermoFisher). Antibodies were eluted in 0.1 M citrate, 0.1 M NaCl, 10%glycerol, 10% sucrose, pH 3.5.

To express bispecific UniAbs, ExpiCHO cells were transfected with twoexpression vectors (knob and hole vectors, knob vectors containC-terminal His-tag) and were expressed in the ExpiCHO cells according tomanufacturer's instructions using the high titer protocol. Clarifiedsupernatants were harvested and the antibodies were purified by IMAC (NiSepharose® Excel, Cytive Life Sciences), using an imidazole gradient forelution. The IL-2Rβγ bispecific UniAbs containing fractions were pooled,concentrated, and further purified on cation exchange to remove anyproduct-related impurities (Mono S® 10/100 GL column (Cytiva LifeSciences)). All antibodies were analyzed by SEC-UPLC and SDS-PAGE toconfirm their size and purity.

The cynomolgus IL-2Rγ sequence was obtained from Uniprot.org (UniProtAccession ID: G7Q2Z6,) and the extracellular domain (aa Met1-Asn254) wascloned into a proprietary vector containing the endogenous leadersequence and a C-terminal His-tag. The IL-2Rγ reagent was expressed inExpiCHO cells, according to the vendors instructions (high titerprotocol, ThermoFisher). Cells were harvested on day 8 and supernatantwas run on SDS-PAGE (NuPAGE 4-12% Bis Tris Gel) to verify target proteinexpression. Clarified harvest was purified by IMAC using Ni-SepharoseExcel resin (Cytiva Life Sciences), using an imidazole gradient forelution. The peaks were pooled and quantified using QiaXpert (Qiagen).

The cloning, expression, and purification of mutant IL-2 protein (T3A,F42A, Y45A, L72G, C125A) was completed at Lake Pharma. A C-terminalHis-tag was added to enable purification by IMAC using standardprocedures and elution with an imidazole gradient.

Octet-Based Off-Rate Measurements

All off-rate measurements were performed on an Octet Qk384 instrument(ForteBio), in 96-well microplates at 25° C. using anti-human IgG Fccapture (AHC, 18-5005) sensors with a shake speed of 1000 rpm. Foroff-rate determination, the antibodies were loaded on the AHC sensors at5 μg/mL. Following a short baseline in kinetics buffer (0.02% Tween20,0.1% BSA, 0.05% sodium azide, 1×PBS). Offrate measurements were done forthe following: human IL-2Rβ (AcroBiosystems), human IL-2Rγ (SinoBiological), cynomolgus IL-2Rβ (Sino Biological), cynomolgus IL-2Rγ(expressed and purified in house using ExpiCHO expression systemfollowed by Ni-NTA His-tag purification), mouse IL-2Rγ (SinoBiological), mouse IL-2Rβ (Sino Biological), human IL-2Rα (SinoBiological), IL-4R (Sino Biological), IL-7R (Sino Biological), IL-9R(R&D Systems) and IL-21R (Sino Biological). The following antibodieswere used as positive controls to verify target binding and reagentquality: anti-human IL-9R (R&D Systems), anti-human IL-21R (R&DSystems), anti-human IL-7R (R&D Systems) and anti-human IL-4Ra (R&DSystems). The loaded sensors were then submerged in wells containingantigen at 100 nM concentration for association step. Dissociation wasmonitored in kinetics buffer. The capture surfaces were regenerated for60 s. ForteBio data analysis software was used to fit the data to a 1:1binding model to extract an association rate and dissociation rate.

Octet-Based Kinetics Measurements

All kinetics measurement experiments were performed on a ForteBio OctetQk384 instrument using anti-human Fc capture (AHC, 18-5005) sensors. Thebispecific UniAbs and antigens were diluted to final concentrations inKinetics buffer (0.02% Tween20, 0.1% BSA, 0.05% sodium azide, 1×PBS).Kinetics measurements were against the following antigens: human IL-2RB(AcroBiosystems), human IL-2RG (AcroBiosystems), cynomolgus IL-2RB (SinoBiological), cynomolgus IL-2RG (expressed and purified in house usingExpiCHO expression system followed by Ni-NTA his-tag purification). Theantibodies were loaded on the AHC sensors at 5 μg/mL for maximumloading. Following a short baseline in Kinetics buffer, the sensors wereexposed to a series of analyte concentrations (7.8 nM to 500 nM) forassociation step and background subtraction was used to correct forsensor drifting. Dissociation was monitored in Kinetics buffer. Thecapture surfaces were regenerated for 60s. All experiments wereperformed with shaking at 1000 rpm. ForteBio's data analysis softwarewas used to fit the data to a 1:1 binding model to extract anassociation rate and dissociation rate. The KD was calculated using theratio kd/ka. Kinetics data for six bispecific antibody constructs isprovided in FIG. 1 .

Biophysical Characterization Assay (Tm, Tagg)

Tm and Tagg were measured on the UNcle platform. Briefly, 9 μL of eachsample was loaded in duplicate in a Uni (UNcle cassette) and run with athermal ramp from 20° C. to 70° C. at a constant rate of 1° C./min.UNcle Analysis 3.1 software, was used to calculate the Tm of each sampleusing the first derivative of the barycentric mean (BCM) of thefluorescence intensity. The Tagg for each sample was calculated usingthe intensity of scattered light at 266 nm.

Thermal Stress and Stability Characterization

Bispecific UniAb molecules were concentrated to 10 mg/mL in 20 mMcitrate and 0.1 M NaCl pH 6.2. Presence of high and low molecular weightspecies (% HMW and % LMW) was determined before and after temperaturestress for 1 month at 2-8° C. and 37° C. by SEC on an analyticalThermoFisher UltiMate™ 3000 UPLC.

Example 1: Identification of IL-2Rβγ Bispecific Antibody Combinationswith Agonist Activity

The activation of the IL-2 receptor complex triggers a signaling cascadethat results in the phosphorylation of STAT5 (pSTAT5), translocation ofpSTAT5 dimers to the nucleus, and transcription of STAT5-regulated genes(M. Rickert, et al., Science 308, 1477-1480 (2005); G. C. Sim, et al.,Cytokine Growth F R 25, 377-390 (2014)). As a primary assay to determineif bispecific antibodies targeting the beta and gamma subunits of IL-2Rcould induce activation of IL-2R signaling, 5 anti-IL-2Rβ binding armsfrom unique CDR3 families and 5 anti-IL-2Rγ binding arms from uniqueCDR3 families were combined to make 25 bispecific UniAbs for conductingan all-by-all screen of agonist activity. The bispecific UniAbs wereexpressed on a silenced and stabilized human IgG4 Fc (CH1 domaindeleted) using knobs-into-holes technology to facilitate heavy-chainheterodimer formation, with a single anti-IL-2Rγ VH on the knob arm anda single anti-IL-2Rβ VH on the hole arm (J. B. B. Ridgway et al.,Protein Eng Des Sel 9, 617-621 (1996); S. M. Canfield, et al., J ExpMedicine 173, 1483-1491 (1991); D. Xu et al., Cell Immunol 200, 16-26(2000); J. W. Bloom et al., Protein Sci 6, 407-415 (1997); M. P. Reddyet al., J Immunol 164, 1925-1933 (2000); A. M. Merchant et al., NatBiotechnol 16, 677-681 (1998)).

A phospho-flow cytometry assay was used to measure and compare thephosphorylation of STAT5 by the 25 IL-2Rβγ UniAb combinations comparedto rhIL-2 on human CD8+ T-cells. STAT5 phosphorylation was not observedwith any of the anti-IL-2Rβ or anti-IL-2Rγ monospecific UniAbs.Similarly, STAT5 phosphorylation was also not observed when anti-IL-2Rβand anti-IL-2Rγ monospecific UniAbs were tested as a mixture in thepSTAT5 assay (FIG. 2B). In contrast, the bispecific UniAbs with oneanti-IL-2Rβ arm and one anti-IL-2Rγ arm exhibited varying levels ofagonist activity, summarized in FIG. 2A. Interestingly, the ability toinduce phosphorylation of STAT5 agonist activity seemed highly dependenton the anti-IL-2Rβ arm present in the bispecific combination, while thedegree of agonism appeared to be dependent on the anti-IL-2Rγ arm.Control data is shown in FIG. 2C.

To identify antibodies with a greater range of agonist activity, asecondary diversity screen was initiated to survey other unique VHsequences in 3 of the 4 lead CDR3 clonotype families identified in thebispecific screen for STAT5 activity. These additional VH sequences wereselected from the lead CDR3 clonotype families and contain sequencevariation in CDR1, CDR2 and framework regions. In total, an additional157 unique family members underwent a second round of high-throughputgene assembly, expression and were assessed for binding to IL-2Rexpressing cells. For IL-2Rβ, an additional 33 IL-2Rβ family F09 membersand an additional 22 IL-2Rβ family F18 members that bound to human andcynomolgus IL-2Rβ cells were identified in the diversity screen. Afurther 29 IL-2R_(y) family F16 members were identified that bound tohuman and cynomolgus IL-2Rγ recombinant protein and on cells in thediversity screen. This large and diverse set of novel IL-2R bindingUniAbs enabled subsequent efforts to identify a set of lead IL-2Rβγbispecific combinations with a range of functional activity.

Example 2: In Vitro Characterization of IL-2Rβγ Bispecific UniAbs

Based on the primary and secondary binding screening results as well asthe STAT5 phosphorylation seen in the all-by-all bispecific UniAbscreen, 6 IL-2Rβγ bispecific UniAb molecules were selected foradditional in vitro characterization. The 6 IL-2Rβγ bispecific UniAbsbound efficiently to both human and cynomolgus T-cells with a range ofEC50 values (FIG. 3 ). None of the 6 bispecific UniAbs bound to othercommon gamma chain partners (IL-4R, IL-7R, IL-9R or IL-21R) or IL-2Rα byOctet off-rate analysis.

The ability of the IL-2Rβγ bispecific UniAbs to stimulate IL-2Rsignaling in human CD4+T, CD8+T, and NK-cells was confirmed by adose-dependent increase of STAT5 phosphorylation compared to rhIL-2 andan rhIL-2 variant which contains mutations (F42A, Y45A, L72G) that havebeen shown to disrupt binding to IL-2Rα while retaining the ability tobind and activate the intermediate affinity IL-2Rβγ receptor (FIGS.4A-4C) (C. Klein et al., Oncoimmunology 6:3 e1277306 (2017)). On CD8+T-cells, the bispecific UniAbs exhibit a range of EC50 values in thepSTAT5 assay, with multiple constructs (BsAb-1, BsAb-3, BsAb-4) showingnear equivalent activity with rhIL-2 and the rhIL-2 variant (FIG. 4A).However, this is in stark contrast to the level of pSTAT5 inCD4+CD25+FoxP3+ T-regulatory cells, where the bispecific UniAbs showsignificantly lower potency compared to rhIL-2 on cells that expresshigh levels of IL-2Rα (FIGS. 4C-4D). Thus, IL-2Rβγ bispecific UniAbsavoid the preferential activation of T-regs, which is a key functionalcriterion for these molecules. All 6 bispecific UniAbs were alsoconfirmed to activate IL-2R signaling on cynomolgus T-cells,establishing cynomolgus monkeys as a suitable non-human primate model insubsequent studies (FIG. 4E).

To further compare the functional activity among the 6 bispecific UniAbsand rhIL-2, a cell proliferation assay was performed. In response totreatment with the bispecific IL-2R agonist UniAbs, or the IL-2 cytokinecontrols, immune effector cells (T and NK-cells derived from healthydonor PBMCs) demonstrated dose dependent proliferation (FIGS. 5A-D).While a range of potencies was observed in the proliferation of CD8+T-cells and NK-cells in PBMCs treated with the IL-2Rβγ bispecificUniAbs, several (BsAb-1, BsAb-3, BsAb-4) showed induction ofproliferation at levels similar to rhIL-2 and the rhIL-2 variantcontrol, while all molecules achieved similar levels of maximumproliferation (FIGS. 5A-5B). In contrast, rhIL-2 was more active thanthe bispecific agonist UniAbs and the rhIL-2 variant control on CD4+cells (including T-regs) (FIGS. 5C-5D).

Cytokine release profiles of the bispecific IL-2R agonist UniAbscompared to rhIL-2 were assessed in an ex vivo human whole blood assay.After a 24-hour incubation in the presence of the IL-2Rβγ bispecificUniAbs or rhIL-2, a dose-dependent increase in IFN-γ, TNF-α, IL-6, andIL-8 was observed for all test articles (FIGS. 6A-6D). Two of thebispecific UniAbs (BsAb-3 and BsAb-4) induced cytokine levels (maxconcentration or EC50) at or above that of rhIL-2 in all testedcytokines, but the remaining four induced levels lower than the cytokinecontrol.

In summary, six bispecific IL-2Rβγ antibodies were identified with arange of agonist activity. BsAb-1 demonstrates agonist activity at asimilar level to that seen with rhIL-2 in immune effector cells measuredby phosphorylation of STAT5 and in the proliferation assay. In contrast,in the same in vitro assays, BsAb-2 shows reduced potency compared torhIL-2 and BsAb-1. Both antibodies showed low aggregation measured bySEC, had favorable melting temperatures, and were stable at 37° C. forone month (FIG. 9 ). These results combined with the favorable cytokinerelease profiles of BsAb-1 and BsAb-2 led to the selection of these twobispecific antibodies for further in vivo characterization.

Example 3: In Vivo Characterization of IL-2Rβγ Bispecific UniAbs BsAb-1and BsAb-2

Prior to conducting in vivo functional studies, the in vivo stabilityand pharmacokinetics of the bispecific antibodies were measured in mice.The observed 5-7 day half-life of each bispecific antibody is consistentwith the half-life of a human IgG4 antibody in mice (FIG. 8A) (R. Denget al., Mabs 3, 61-66 (2011)). To assess the in vivo functional activityof the bispecific antibodies, an accelerated graph versus host disease(GVHD) model was used to compare the functional activity of BsAb-1,BsAb-2 and rhIL-2 (FIGS. 10A-10C). In the first experiment, irradiatedNSG mice were engrafted with human PBMCs, and the mice were subsequentlytreated with either vehicle, rhIL-2 daily, or one of the two bispecificagonist antibodies twice a week until sacrifice. As expected, animalstreated with the vehicle control showed onset of GVHD, measured by bodyweight loss, around day 20 and were sacrificed with 20% body weight lossat approximately day 35. In contrast, the bispecific IL-2Rβγ agonistantibodies (BsAb-1, BsAb-2) as well as rhIL-2-treated animals exhibitedonset of GVHD at approximately day 8 and were sacrificed with 20% bodyweight loss between days 9 and 13, indicating an acceleration of GVHDcompared to the vehicle control, consistent with the enhanced activationof immune effector cells in treated mice (FIG. 10B).

A second study was conducted to directly measure the ability of BsAb-1and BsAb-2 to stimulate the proliferation of immune effector cells invivo. Similar to the first experiment, irradiated NSG mice wereengrafted with human PBMCs that were labeled with CSFE and treated withvehicle, rhIL-2, BsAb-1, or BsAb-2. After day 5 of treatment, spleenswere harvested and the proliferation of CD8+T and CD4+ T-cells wascompared between the 4 treatment groups by measuring CSFE staining inthe different lymphocyte populations. BsAb-1 and BsAb-2 both showedsignificantly more proliferating CD8+ T-cells compared to rhIL-2 and thevehicle control (FIG. 10C). CD4+ T-cells were expanded to a lesserextent; however, a significant increase in proliferating CD4+ T-cellswas seen in BsAb-2 treated mice compared to the vehicle control.

An important aspect of the preclinical evaluation of the bispecificantibody agonists was establishing cynomolgus monkeys as an appropriatein vivo model for measuring the pharmacodynamics of the molecules. Todetermine human and cynomolgus functional equivalency, the bispecificantibodies were confirmed to activate pSTAT5 signaling ex vivo incynomolgus primary T-cells at a similar level as seen in primary humanT-cells (FIG. 4C and FIG. 4E). After establishing functional equivalencybetween human and cynomolgus, a non-GLP cynomolgus study was conductedto further investigate the activity of BsAb-1 and BsAb-2 in vivo in anon-human primate model. The two bispecific agonist antibodies wereadministered to cynomolgus monkeys in groups of 2 that received a singleintravenous (slow bolus) dose of 0.03, 0.1 or 0.3 mg/kg of either BsAb-1or BsAb-2. At all doses with both molecules, a marked expansion ofperipheral CD8+T and NK-cells was observed (FIGS. 11A-11K). After aninitial transient drop in lymphocyte numbers, CD8+T, NK-cells, and to alesser degree, CD4+ T-cells, showed dose dependent proliferation andexpansion in the blood, peaking around day 4-7 before returning tobaseline levels around day 14 (FIGS. 11A-11C and 11F-11H). Importantly,no pronounced expansion of CD4+CD25+FoxP3+ T-regulatory cells was seen,consistent with the bispecific agonist antibodies avoiding preferentialactivation of the trimeric IL-2 receptor (FIG. 11D and FIG. 11I). Thiseffect was further confirmed by the ratio of CD8+:CD4+ T-cells which wasskewed in favor of the CD8+ T-cell subset (FIG. 11K). Moreover, theIL-2Rβγ agonist antibodies were well tolerated in the monkeys at alldose levels tested, with no indication of vascular leak syndrome orother overt toxicities.

Example 4: In Vivo Characterization of IL-2Rβγ Bispecific UniAb BsAb-5

Prior to conducting in vivo functional studies, the in vivo stabilityand pharmacokinetics of the bispecific antibodies were measured in miceat two dose-levels: 1 mg/kg and 10 mg/kg. The observed 5-day half-lifeof BsAb-5 (IL2RB_F09K**IL2RG_F16B) is consistent with the half-life of ahuman IgG4 antibody in mice (FIGS. 12A-12B) (R. Deng et al., Mabs 3,61-66 (2011)). To assess the in vivo functional activity of thebispecific antibody, an accelerated graph versus host disease (GVHD)model was used to compare the functional activity of BsAb-5 and rhIL-2(FIGS. 13-14 ). In the first experiment, irradiated NSG mice wereengrafted with human PBMCs, and the mice were subsequently treated witheither vehicle, rhIL-2 daily, or BsAb-5 twice a week until sacrifice. Asexpected, animals treated with the vehicle control showed onset of GVHD,measured by body weight loss, around day 20 and were sacrificed with 20%body weight loss at approximately day 35. In contrast, BsAb-5-treatedanimals, as well as rhIL-2-treated animals, exhibited onset of GVHD atapproximately day 8 and were sacrificed with 20% body weight lossbetween days 9 and 13, indicating an acceleration of GVHD compared tothe vehicle control, consistent with the enhanced activation of immuneeffector cells in treated mice (FIG. 14 ).

A second study was conducted to directly measure the ability of BsAb-5to stimulate the proliferation of immune effector cells in vivo. Similarto the first experiment, irradiated NSG mice were engrafted with humanPBMCs that were labeled with CPD450 and treated with vehicle, rhIL-2, orBsAb-5. After day 5 of treatment, spleens were harvested and theproliferation of CD8+ T-cells, CD4+ T-cells, and NK-cells were comparedbetween the 3 treatment groups by measuring CPD450 dilutions in thedifferent populations. In all three measured cell types, BsAb-5 inducedsignificantly more proliferation than rhIL-2 or the vehicle control(FIGS. 15A-15C).

An important aspect of the preclinical evaluation of the bispecificantibody agonists was establishing cynomolgus monkeys as an appropriatein vivo model for measuring the pharmacodynamics of the molecules. Todetermine human and cynomolgus functional equivalency, the bispecificantibodies were confirmed to activate pSTAT5 signaling ex vivo incynomolgus primary T-cells at a similar level as seen in primary humanT-cells (FIG. 4C and FIG. 4E). After establishing functional equivalencybetween human and cynomolgus, a non-GLP cynomolgus study was conductedto further investigate the activity of BsAb-5 in vivo in a non-humanprimate model. BsAb-5 was administered to cynomolgus monkeys in a singleintravenous (slow bolus) dose of 0.1, 0.3, or 0.5 mg/kg. The monkeyswere dosed in groups of 2 for the first two dose levels, and in a groupof 4 for the 0.5 mg/kg dose level. At all dose levels, a markedexpansion of peripheral CD8+ T-, NK-, and NKT-cells was observed (FIGS.16A-16F). After an initial transient drop in lymphocyte numbers, CD8+T-, NK-, NKT-cells, and to a lesser degree, CD4+ T-cells, showed dosedependent proliferation and expansion in the blood, peaking around day4-7 before returning to baseline levels around day 14 (FIGS. 16A-16H).Importantly, no preferential expansion of CD4+CD25+Foxp3+ T-regulatorycells was observed, consistent with the bispecific agonist antibodiesavoiding preferential activation of the trimeric IL-2 receptor (FIGS.16A-16J). Additionally, B-cells, which serve as a useful negativecontrol due to their lack of IL-2 receptor expression, did notproliferate in response to BsAb-5 (FIGS. 16K-16L). Moreover, the IL-2Rβγagonist antibodies were well tolerated in the monkeys up to 0.5 mg/kg,with no indication of vascular leak syndrome or other overt toxicities.

While non-limiting example embodiments of the present disclosure havebeen shown and described herein, it will be obvious to those skilled inthe art that such embodiments are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

The invention claimed is:
 1. A heavy chain-only antibody comprising: afirst heavy chain variable region that binds to IL2RB, comprising: aCDR1 sequence comprising the formula: G G S I S S S X1 W (SEQ ID NO: 26)wherein X1 is D or N; and a CDR2 sequence comprising the formula: I X2 HS G S T (SEQ ID NO: 27) wherein X2 is D or S; and a CDR3 sequencecomprising the formula: X3 R G X4 W E L X5 D A F D I (SEQ ID NO: 28)wherein X3 is G or A; X4 is S or Q; and X5 is S or T; and a second heavychain variable region that binds to IL2RG, comprising: a CDR1 sequencecomprising the formula: G F X1 X2 X3 X4 Y Y (SEQ ID NO: 32) wherein X1is T or I; X2 is F or V; X3 is S, N, or G; and X4 is D or N; and a CDR2sequence comprising the formula: I S X5 S G X6 X7 I (SEQ ID NO: 33)wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and a CDR3sequence comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20).
 2. Theheavy chain-only antibody of claim 1, wherein: the first heavy chainvariable region comprises: (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 7; or (b) aCDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 4, and aCDR3 sequence of SEQ ID NO: 8; or (c) a CDR1 sequence of SEQ ID NO: 2, aCDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 9; andthe second heavy chain variable region comprises: (a) a CDR1 sequence ofSEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 17, and a CDR3 sequence ofSEQ ID NO: 20; or (b) a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequenceof SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 20; or (c) a CDR1sequence of SEQ ID NO: 16, a CDR2 sequence of SEQ ID NO: 18, and a CDR3sequence of SEQ ID NO:
 20. 3. The heavy chain-only antibody of claim 1,wherein the first heavy chain variable region has at least 95% sequenceidentity to any one of SEQ ID NOs: 11-13.
 4. The heavy chain-onlyantibody of claim 1, wherein the first heavy chain variable regionsequence is selected from SEQ ID NOs: 11-13.
 5. The heavy chain-onlyantibody of claim 1, wherein the second heavy chain variable region hasat least 95% sequence identity to any one of SEQ ID NOs: 22-24.
 6. Theheavy chain-only antibody of claim 1, wherein the second heavy chainvariable region sequence is selected from SEQ ID NOs: 22-24.
 7. Theheavy chain-only antibody of claim 1, wherein: the first heavy chainvariable region comprises a CDR1 sequence of SEQ ID NO: 1, a CDR2sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 7; and thesecond heavy chain variable region comprises a CDR1 sequence of SEQ IDNO: 15, a CDR2 sequence of SEQ ID NO: 17, and a CDR3 sequence of SEQ IDNO:
 20. 8. The heavy chain-only antibody of claim 7, wherein the firstheavy chain variable region has at least 95% sequence identity to SEQ IDNO: 11, and the second heavy chain variable region has at least 95%sequence identity to SEQ ID NO:
 22. 9. The heavy chain-only antibody ofclaim 7, wherein the first heavy chain variable region comprises SEQ IDNO: 11, and the second heavy chain variable region comprises SEQ ID NO:22.
 10. The heavy chain-only antibody of claim 7, comprising a firstpolypeptide comprising SEQ ID NO: 53 and a second polypeptide comprisingSEQ ID NO:
 61. 11. The heavy chain-only antibody of claim 1, wherein:the first heavy chain variable region comprises a CDR1 sequence of SEQID NO: 1, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ IDNO: 8; and the second heavy chain variable region comprises a CDR1sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 18, and a CDR3sequence of SEQ ID NO:
 20. 12. The heavy chain-only antibody of claim11, wherein the first heavy chain variable region has at least 95%sequence identity to SEQ ID NO: 12, and the second heavy chain variableregion has at least 95% sequence identity to SEQ ID NO:
 23. 13. Theheavy chain-only antibody of claim 11, wherein the first heavy chainvariable region comprises SEQ ID NO: 12, and the second heavy chainvariable region comprises SEQ ID NO:
 23. 14. The heavy chain-onlyantibody of claim 11, comprising a first polypeptide comprising SEQ IDNO: 62 and a second polypeptide comprising SEQ ID NO:
 63. 15. The heavychain-only antibody of claim 1, wherein: the first heavy chain variableregion comprises a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of anda CDR3 sequence of SEQ ID NO: 9, and the second heavy chain variableregion comprises a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence ofSEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO:
 20. 16. The heavychain-only antibody of claim 15, wherein the first heavy chain variableregion has at least 95% sequence identity to SEQ ID NO: 13, and thesecond heavy chain variable region has at least 95% sequence identity toSEQ ID NO:
 23. 17. The heavy chain-only antibody of claim 15, whereinthe first heavy chain variable region comprises SEQ ID NO: 13, and thesecond heavy chain variable region comprises SEQ ID NO:
 23. 18. Theheavy chain-only antibody of claim 15, comprising a first polypeptidecomprising SEQ ID NO: 64, and a second polypeptide comprising SEQ ID NO:65.
 19. The heavy chain-only antibody of claim 1, wherein: the firstheavy chain variable region comprises a CDR1 sequence of SEQ ID NO: 1, aCDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8, andthe second heavy chain variable region comprises a CDR1 sequence of SEQID NO: 16, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQID NO:
 20. 20. The heavy chain-only antibody of claim 19, wherein thefirst heavy chain variable region has at least 95% sequence identity toSEQ ID NO: 12, and the second heavy chain variable region has at least95% sequence identity to SEQ ID NO:
 24. 21. The heavy chain-onlyantibody of claim 19, wherein the first heavy chain variable regioncomprises SEQ ID NO: 12, and the second heavy chain variable regioncomprises SEQ ID NO:
 24. 22. The heavy chain-only antibody of claim 19,comprising a first polypeptide comprising SEQ ID NO: 34 and a secondpolypeptide comprising SEQ ID NO:
 35. 23. The heavy chain-only antibodyof claim 1, wherein: the CDR1, CDR2, and CDR3 sequences in the firstheavy chain variable region are present in a human VH framework; and/orthe CDR1, CDR2, and CDR3 sequences in the second heavy chain variableregion are present in a human VH framework.
 24. The heavy chain-onlyantibody of claim 1, comprising a heavy chain constant region comprisinga hinge region, a CH2 domain, and a CH3 domain, wherein the heavy chainconstant region does not contain a CH1 sequence.
 25. A pharmaceuticalcomposition comprising: a heavy chain-only antibody of claim 1; and apharmaceutically acceptable excipient.